THE  LIBRARY 

OF 

THE  UNIVERSITY 
OF  CALIFORNIA 

PRESENTED  BY 

PROF.  CHARLES  A.  KOFOID  AND 
MRS.  PRUDENCE  W.  KOFOID 


ipf 


<! 


JUL 


THE 

TRANSMUTATION  OF  BACTERIA 


CAMBRIDGE  UNIVERSITY  PRESS 

C.  F.  CLAY,  MANAGER 
LONDON   :   FETTER  LANE,  E.G.  4 

LONDON  :   H.  K.  LEWIS  AND  CO.,  LTD., 
136  Gower  Street,  W.C.  i 

LONDON  :  WILLIAM  WESLEY  AND  SON; 
28  Essex  Street,  Strand,  W.C.  2 

NEW  YORK    :    G.  P.  PUTNAM'S  SONS 

BOMBAY      ) 

CALCUTTA}.  MACMILLAN  AND  CO.,  LTD. 

MADRAS     j 

TORONTO  :  J.  M.  DENT  AND  SONS,  LTD. 

TOKYO  :  MARUZEN-KABUSHIKI-KAISHA 

ALL  RIGHTS  RESERVED 


THE 

TKANSMUTATION  OF  BACTEKIA 


BY 
8.  GURNEY-DIXON,  M.A.,  M.D.  (CANTAB.) 

M.R.C.S.  (BNG.),  L.R.C.P.  (LOND.) 


"Ogni  primaio  aspetto  ivi  era  casso: 
due  e  nessun  V  imagine  perversa 
parea,... 
Cosi  vid'  io  la  settima  zavorra 

mutare  e  trasmutare;  e  qui  mi  scusi 
la  novita,  se  fior  la  penna  abborra." 
Dante:  Inf.  XXV. 


CAMBRIDGE 

AT  THE  UNIVERSITY  PRESS 
1919 


Ulb. 

PREFACE 

r  I  THIS  essay  is  based  upon  notes  and  observations  which  I 
-"-  collected  previous  to  the  year  1913.  It  was  only  partly 
written  when,  in  August  1914,  I  proceeded  on  active  service. 
I  was  able,  however,  to  complete  it  in  the  following  summer 
while  serving  with  a  Field  Ambulance  in  France,  and  in  the 
autumn  of  the  same  year  (1915)  I  submitted  it  in  the  form  of 
a  Dissertation  for  the  degree  of  M.D.  at  the  University  of 
Cambridge. 

The  difficulties  in  carrying  out  work  of  this  character 
while  serving  at  the  Front — remote  from  libraries  and  amidst 
"  alarms  and  excursions  "  which  break  up  one's  scanty  leisure 
— are  sufficiently  obvious  and  I  trust  may  excuse  some 
of  its  defects.  Some  valuable  materials  which  I  had  hoped  to 
utilise,  including  chapters  on  Viability  and  Agglutination 
Reactions,  were  buried  by  a  shell  explosion  and  could  not 
be  replaced. 

The  claims  of  Army  work  have  also  precluded  any  attempt 
on  my  part  to  bring  the  work  up  to  date  by  reference  to 
papers  published  since  the  beginning  of  the  war.  I  particu- 
larly regret  having  learnt  too  late  to  include  any  mentio'n 
of  it  in  the  following  pages  of  the  valuable  research  carried 
out  by  Dr  Thiele  and  Dr  Embleton  on  the  part  played  by 
the  body  ferments  in  the  pathogenicity  of  bacteria. 

Though  I  have  endeavoured  to  suppress  all  irrelevant 
matter,  I  am  only  too  conscious  of  the  discursiveness  of  this 
essay.  The  topic  is  one  of  absorbing  interest  and  at  every  step 
one  is  tempted  to  digress.  In  the  words  of  Dante,  which  I 
have  quoted  on  the  title  page,  "The  novelty  must  be  my 
excuse  if  my  pen  has  wandered  at  all/' 


vi  PREFACE 

I  have  only  touched  the  fringe  of  the  subject.  An  inex- 
perienced sailor  in  my  "  piccioletta  barca,"  I  have  been  tossed 
about  in  the  breakers  of  this  uncrossed  sea.  To  others,  better 
equipped  by  knowledge  arid  training  than  I  am  to  explore  it, 

I  would  say — 

"  Metier  potete  ben  per  1'  alto  sale 

vostro  navigio,... 
Quei  gloriosi  che  passaro  a  Colco 
non  s'  ammiraron,  come  voi  farete, 
quando  Jason  vider  fatto  bifolco." 

(Dante :  Par.  II.) 

S.  G.-D. 


CONTENTS 


PAGE 

PREFACE    v 

SYNOPSIS ix 

INTRODUCTION 1 

CHAP. 

*  I.        THE  SCOPE  OF  THE  ENQUIRY        ...  3 

II.  CONDITIONS  MODIFYING  THE  CHARACTERS 

OF  BACTERIA 13 

III.  A  CONSIDERATION  OF  THE  EVIDENCE       .  28 

IV.  VARIATIONS  IN  MORPHOLOGY      ...  37 

V.  VARIATIONS  IN  FERMENTING  POWER       .  50 

VI.  VARIATIONS  IN  VIRULENCE  .        .        .        .  71 

VII.  VARIATIONS  IN  PATHOGENIClTY         .    .    .  94 

VIII.  THE    POSSIBLE    OCCURRENCE   OF   TRANS- 
MUTATION IN  THE  LIVING  BODY         .        .  107 

IX.      SUPPOSED  INSTANCES  OF  TRANSMUTATION 

BROUGHT  ABOUT  EXPERIMENTALLY  .        .116 

X.       SUMMARY 140 

XL      THE  ENZYME  THEORY  OF  DISEASE     .        .  153 

XII.     CONCLUSIONS 170 

APPENDIX.    REFERENCES    .  171 


ao 


SYNOPSIS 


INTRODUCTION 
CHAPTER  I 

THE  SCOPE  OF  THE  ENQUIRY 

DEFINITION  OF  TERMS.  Transmutation  not  evolution — evolution  in  bac- 
teria— its  stages.  Natural  variation — "Spontaneous"  and  "impressed."  Varia- 
tion easily  studied  in  bacteria — unicellular  organisms — method  of  generation 
— rapidity  of  generation — environment  easily  modified.  Natural  selection. 
Artificial  selection.  "Transmutation  of  Species"  apparently  contradictory — 
meaning  of  "species" — based  on  characters.  Arbitrary  nature  of  distinction 
between  species — illustrated  by  streptococci — classified  according  to  food- 
stuffs and  haemolytic  power,  adhesiveness,  staining,  cultural  characters, 
virulence  and  pathogenicity,  agglutination,  fermenting  power.  "Species" 
not  a  rigid  term. 

A  CONSIDERATION  OF  THE  POSSIBILITIES.  1.  Simple  variation.  2.  Varia- 
tions in  different  directions  associated.  3.  Development  of  intermediate 
forms.  4.  Slight  changes  in  closely  allied  organisms.  5.  Complete  change  in 
characters.  (Pages  3 — 12) 

CHAPTER  II 

CONDITIONS  MODIFYING  THE  CHARACTERS  OF  BACTERIA 

1.  Spontaneous  variations.  Pleomorphism.  Unexplained  variations. 
2.  Geographical  distribution.  3.  Prolonged  cultivation — extends  survey — 
permits  natural  selection — influence  of  saprophytism.  4.  Conditions  of  culti- 
vation, (a)  lowered  vitality,  (6)  crowding  of  colonies,  (c)  temperature,  (d)  at- 
mospheric pressure,  (e)  oxygen,  (/)  sunlight.  5.  Ultra  violet  rays.  6.  Elec- 
trolysis. 7.  Age  of  culture — pleomorphism — other  variations.  8.  Culture 
medium— (a)  age  of  medium,  (&)  reaction  of  medium,  (c)  nature  of  medium 
—natural  secretions— pathological  exudations— water,  (d)  chemical  sub- 
stances—carbolic acid,  antiseptics,  boric  acid,  potassium  bichromate,  sodium 
benzoate,  glycerine,  iodine  trichloride,  lactic  acid.  9.  Prolonged  contact 
with  particular  foodstuff.  10.  Artificial  selection— method  sometimes  in- 
effective. 11.  Symbiosis— lichens— nitrifying  organisms— parasitism— anaer- 
obes. Symbiosis  may  confer  new  powers — may  have  no  effect.  Methods  of 
studying  symbiosis— mixed  growth,  adjacent  colonies,  criss-cross  planting, 
surface  and  deep  growths,  double  celluloid  sac,  successive  growth.  12.  Para- 
sitism, (a)  transmission  through  alimentary  canal,  (6)  passage,  (c)  celioidin 
sac  in  body  cavity,  (d)  residence  in  living  tissues,  (e)  during  disease,  (/)  in 
"carriers."  (13—27) 


x  SYNOPSIS 

CHAPTER  III 

A  CONSIDERATION  OF  THE  EVIDENCE 

1.  Contamination.  Growth  from  single  bacterium.  2.  Mixed  infection — 
error  due  to  (a)  unequal  growth  of  two  strains,  (6)  incomplete  recognition. 
Proof  of  continuity  necessary.  3.  Secondary  invasion.  Bacteria  in  healthy 
organs.  Post  mortem  invasion.  4.  Repetition  of  experiment.  5.  Constancy 
of  new  feature— meaning  of  "permanent."  6.  Perseverance  necessary. 
7.  Faultless  technique  (e.g.  agglutination)  and  accurate  observation  (e.g. 
staining)  required.  8.  Methods  may  require  to  be  improved,  (a)  irregular 
results  due  to  media — e.g.  sugars,  may  be  impure,  contaminated  by  glass, 
affected  by  sterilisation,  deteriorate — age  of  medium — composition — re- 
action, (b)  age  of  culture,  (c)  time  allowance.  9.  Clinical  observation  im- 
portant, e.g.  Widal's  test  in  jaundice— effect  of  drugs— pre-existing  disease. 

(28—36) 


CHAPTER  IV 

VARIATIONS  IN  MORPHOLOGY 

A.  ZOOGLEIC  FORMS— not  fortuitous— B.  radicicola—Beggiatoa  versatilis. 
Zoogleae  not  in  strict  sense  individuals— analogy  of  regiment  of  soldiers  and 
crowd  of  pitmen— typical  formations  assumed— not  separate  individuals  like 
a  tree — formations  not  invariable — may  simulate  each  other — this  does  not 
imply  transmutation.     I.   Zoogleic  forms  occurring  spontaneously — stages 
in  life  history — or  variations.   B.  rubescens — other  examples.     II.  Zoogleic 
forms  artificially  produced,  1.   Due  to  chemical  substances — salt,  sewage, 
urea,  saliva,  bile,  acid,  caustic  soda,   /3  naphthol,  alcohol,  potassium  bi- 
chromate, boric  acid,  nitrates,  antiseptics,  tartaric  acid.    2.   Temperature. 
3.  Absence  of  oxygen.    4.   Ultra  violet  rays.    5.   Growth  in  animal  body. 

B.  VARIATIONS  IN  INDIVIDUAL  ORGANISMS.    I.  Pleomorphism — B.  ru- 
bescens— other  examples.  1 1.  Variations  due  to  environment.  1 .  Geographical 
distribution.   2.  Prolonged  cultivation.   3.  Crowding  of  colonies.   4.  Changes 
in  medium — reaction.    5.   Chemical  substances — urea,  urine,  carbolic  acid, 
creosote,  nitrogenous  substances.    6.    Ultra  violet  rays.     7.    Electrolysis. 
8.   Symbiosis.    9.  Growth  in  living  tissues. 

C.  VARIATIONS  IN  COLONIES.    1.   Colonies  of  the  same  organism  vary. 
2.    Different  organisms  produce  similar  colonies.    3.    Addition  of  various 
substances  to  medium  affects  colonies.    4.   Influence  of  heat.    5.   Effect  of 
"passage." 

Variation  in  other  morphological  characters.  (37 — 49) 


SYNOPSIS  xi 

CHAPTER  V 

VARIATIONS  IN  FERMENTING  POWER 

THE  FERMENTATION  OF  CARBOHYDRATES— its  stages.  Different  types  of 
variation.  I.  Different  strains  may  possess  different  fermenting  properties. 
II.  The  same  strain  may  vary  spontaneously.  III.  Fermenting  properties 
modified  by  conditions  of  growth.  1.  Temperature.  2.  Oxygen.  3.  Atmo- 
spheric pressure.  4.  Age  of  culture.  5.  Age  of  medium.  6.  Composition 
of  medium — effect  of  carbolic  acid,  sodium  benzoate,  monochloracetic  acid. 
7.  Influence  of  source — milk,  urine,  ascitic  fluid.  IV.  Symbiosis.  V.  In 
"carriers."  VI.  After  "passage."  VII.  In  disease.  VIII.  Prolonged  con- 
tact with  a  particular  sugar.  IX.  Artificial  selection — method  often  in- 
effective. 

THE  SIGNIFICANCE  OF  VARIATIONS  IN  SUGAR  REACTIONS.  1.  Fermentation 
due  to  enzymes  which  are  destroyed  by  antiseptics.  2.  Distinct  enzyme  for 
each  different  sugar.  3.  Different  enzyme  for  each  different  stage  in  fer- 
mentation. 4.  Distinct  enzyme  for  forming  each  acid.  5.  Distinct  enzyme 
for  producing  gas  from  each  acid.  6.  New  fermenting  power  an  adaptation 
to  environment.  7.  Such  adaptation  advantageous  to  organism.  8.  En- 
couraged by  natural  selection.  9.  Explanation  of  incubation  period — oc- 
cupied by  preparatory  changes  ? — this  disproved, — interval  before  variation 
appears? — this  disproved,— time  required  for  variants  to  predominate? — 
does  not  explain  definite  length  of  period.  10.  Reason  for  shortening  of 
period  by  subculture — subculture  hastens  reproduction.  11.  Artificial 
selection.  12.  Reversion.  13.  Variations  apparently  spontaneous — possibly 
due  to  contamination  of  medium — or  to  impure  sugar.  No  explanation  of 
spontaneous  variation. 

THE  VALUE  OF  THE  SUGAR  REACTIONS — unsatisfactory  as  tests.  1.  Time 
allowance  not  fixed.  2.  Reactions  vary  with  temperature  and  other  con- 
ditions. 3.  Media  often  unreliable — sugars  impure — altered  by  sterilisation 
— contaminated  by  glass  vessel — deteriorate  on  keeping — acid  reaction 
masked.  4.  Tests  inconstant.  5.  Positive  or  negative  reaction  a  matter  of 
degree  only.  6.  Different  carbohydrate  groups  yield  different  classification 
— if  designed  to  correspond  with  other  tests  useful  for  identification  only. 
Comparison  between  fermentation  and  agglutination  tests — fermentation 
tests  may  vary  while  agglutination  constant — fermentation  and  agglutination 
properties  may  both  be  altered — they  yield  a  different  classification — two 
tests  not  related  but  may  supplement  each  other. 

THE  VALUE  OF  VARIATIONS  IN  THE  SUGAR  REACTIONS  IN  THE  IDENTIFICA- 
TION OF  BACTERIA — variations  themselves  constitute  a  test — may  be  specific 
(cf.  morphology  of  B.  diph.} — may  identify  source  of  strain.  (50 — 70) 


xii  SYNOPSIS 

CHAPTER  VI 
VARIATIONS  IN  VIRULENCE 

Bacteria  pathogenic  and  non-pathogenic.  Pathogenic  character  due  to 
two  factors — parasitism — nature  of  activity  in  tissues.  Most  bacteria  cannot 
invade  tissues — activities  of  some  invaders  harmless — actual  invasion  not 
essential.  Effects  of  bacterial  invasion  due  to  (a)  their  metabolism,  (ft)  their 
disintegration,  (c)  their  mechanical  action,  (d]  response  of  living  tissues. 
Viability — pathogenesis — virulence. 

•VARIATIONS  IN  .VIRULENCE.  1.  At  different  stages  of  epidemic— possibly 
explained  by  unequal  resistance  met  with.  2.  Sporadic  cases  of  infectious 
disease  imply  weakened  virulence.  3.  Endemic  diseases  become  less  viru- 
lent— possibly  explained  by  acquired  immunity.  4.  Epidemics  vary  in 
severity  with  date  and  locality.  5.  Intensity  of  infection  by  same  specific 
organism  varies.  6.  Virulence  altered  by  abnormal  conditions  of  cultiva- 
tion, (a)  temperature — possibly  protective  influence  of  fever — disproved, 
(b)  presence  of  antiseptics— carbolic  acid,  potassium  bichromate,  iodine  tri- 
chloride, saliva,  (c)  oxygen,  (d]  sunlight,  (e)  reaction  of  medium.  7.  Virulence 
altered  by  prolonged  cultivation  outside  the  body.  Results  due  to  several 
factors,  (a)  chemical  composition  of  media — blood  media — pathological  exu- 
dations— urine,  (&)  physical  character  of  artificial  media,  (c)  response  of 
tissues,  (d}  purity  of  culture.  8.  Virulence  increased  by  growth  in  patho- 
logical secretions.  9.  Symbiosis — affects  viability — also  affects  virulence. 
10.  Virulence  altered  by  "passage" — passage  alternating  with  culture  more 
effective.  1 1.  Simultaneous  inoculation  with  another  organism  intensifies 
results — even  when  symbiosis  of  same  organism  outside  the  body  ineffective. 
Simultaneous  subcutaneous  and  sub-peritoneal  inoculations  with  different 
organisms  also  effective.  Exalted  virulence  is  towards  species  used  for 
passage — not  necessarily  towards  others. 

THE  SIGNIFICANCE  OP  VARIATION  IN  VIRULENCE.  Evolution  of  bacteria 
— virulence  is  latest  property  acquired  and  first  to  be  lost.  Its  re-acquire- 
ment an  example  of  the  survival  of  the  fittest  —"fittest"  not  necessarily  most 
robust,  but  most  capable  of  defence.  Virulence  results  from  adaptation 
and  is  not  due  to  increased  robustness,  (a)  increased  virulence  to  one  species 
of  animal  does  not  apply  to  another,  (b)  most  virulent  not  always  most  robust 
— contrary  true  of  pneumococcus,  (c)  analogy  suggests  adaptation,  e.g. 
increased  resistance  to  antiseptics,  (d)  increased  virulence  accompanied  by 
other  changes  obviously  adaptive,  e.g.  growth  at  body  temperature.  Diffi- 
culties in  accepting  natural  selection  as  developing  virulence,  (a)  Intra- 
cellular  toxins  only  set  free  after  death  of  organism — may  nevertheless  be 
of  advantage  to  strain — their  effect  perhaps  purely  physiological  and  not 
the  result  of  adaptation,  (b)  Why  are  common  infective  diseases  not  of 
deadly  virulence  ? — death  of  host  involves  death  of  organism,  (c)  Virulence 
established  by  single  "passage" — virulence  possibly  results  from  sudden 
change  in  metabolism,  (d)  Toxic  saprophyte  assists  non-toxic  as  well  as 


SYNOPSIS  xiii 

« 

itself— nevertheless  may  benefit  strain.  Invasion  of  tissues  by  virulent 
saprophyte  involves  change  in  foodstu/s— relationship  between  altered 
metabolism  and  acquirement  of  toxicity — possibly  a  change  in  excretion 
following  change  in  assimilation — experimental  evidence,  (a)  B.  coli  does  not 
attack  proteid  if  carbohydrate  present,  (6)  B.  diph.  does  not  yield  toxin  if 
much  carbohydrate  present — suggest  toxins  may  result  from  alteration  in 
food  material.  Altered  metabolism  of  saprophyte  facilitates  invasion  of 
tissues — this  supposed  alteration  in  metabolism  does  not  always  confer 
toxicity — toxins  may  be  regarded  as  an  excretion  or  as  a  secretion — or  as 
product  of  enzyme— activity  of  enzyme  may  be  due  to  adaptation,  encouraged 
by  natural  selection. 

THE  VALUE  OF  VIRULENCE  IN  CLASSIFICATION.  Classification  according  to 
virulence  inconsistent.  Non-virulent  B.  diph.  in  "carriers"  regarded  as 
lineal  descendant  of  virulent  Klebs-Loeffler  bacillus — other  non-virulent 
B.  diph.  provoke  antitoxin,  therefore  same  species  as  Klebs-Loeffler  bacillus. 
Non-virulent  and  virulent  pneumococcus  regarded  as  varieties  of  same 
species.  Non-virulent  and  virulent  B.  coli  communis  thought  by  some  to  be 
different  species — cf.  amoeba  coli.  Non-virulent  "B.anthracoides"  described 
as  different  species  from  virulent  B.  anthracfs.  S.  erysipelatis  and  S. 
pyogenes  formerly  regarded  as  distinct  species.  Virulence  not  a  specific 
character.  (71—93) 

CHAPTER  VII 

VARIATIONS  IN  PATHOGENICITY 

Pathogenicity  is  power  to  produce  in  certain  animals  certain  symptoms 
and  certain  lesions — quite  distinct  from  virulence  and  other  characters. 
Generally  regarded  as  more  fixed  than  other  characters — constitutes  final 
appeal  in  doubtful  cases,  e.g.  Hofmann's  bacillus  and  Klebs-Loeffler  bacillus 
— gonococcus  and  meningococcus — gonococcus  does  not  cause  meningitis 
nor  meningococcus  urethritis.  Pathogenicity  a  variable  character  in  all 
three  aspects. 

I.  VARIATION  IN  KIND  OF  ANIMAL  AFFECTED. 

II.  VARIATION  IN  SYMPTOMS  CAUSED. 

(1)  Same   organism    causes    different    symptoms   in    different    cases. 
Symptoms  may  depend  upon  organs  affected — cf.  lead  poisoning — this 
determined  by  route  of  infection  and  vitality  of  organs — also  by  patho- 
genicity  of  organism — e.g.  tubercle  bacillus  causes  phthisis,  osteitis,  arthritis, 
lupus — unlike  lead  poisoning  types  remain  distinct — skin  rarely  infected  by 
tuberculous  sputum — contrast  between  gonococcus  and  meningococcus  no 
greater  than  between  different  strains  of  tubercle  bacilli.    Fallacy  due  to 
pre-existing  disease,  e.g.  nephritis  in  cerebrospinal  fever. 

(2)  Pathogenicity  can  be  artificially  modified,  e.g.  that  of  B.  anthracis 
by  ultra  violet  rays. 

(3)  During    epidemic    different    cases    exhibit    different    symptoms. 


xiv  SYNOPSIS 

• 

S.  scarlatinae  causes  scarlet  fever  in  some  cases  and  puerperal  fever  in 
others.  M.  catarrhalis  produces  symptoms  of  many  diseases — common  cold, 
influenza,  scarlet  fever,  diphtheria,  typhoid  fever,  cerebro-spinal  fever. 

(4)  In    different  epidemics   different   types    of   disease  presented — 
B.  influenzas  causes  epidemics  simulating  coryza,  rheumatic  fever,  typhoid 
fever,  cerebrospinal  fever. 

(5)  Same  train  of  symptoms  follows  infection  by  different  organisms 
— typical  rabies  due  to  B.  diph. — typical  scarlet  fever,  cerebrospinal  fever 
and  influenza  due  to  M.  catarrhalis— typical  cerebrospinal  fever  due  to 
B.  typhosus  and  to  Klebs-Loeffler  bacillus— symptoms  resembling  diphtheria 
due  to  pneumococcus — typical  typhoid  fever  due  to  B.  coli. 

III.  VARIATION  IN  LESIONS  PRODUCED — studied  in  two  ways — lesions  pro- 
duced during  disease  and  by  artificial  inoculation  in  animals.  1.  Variations 
in  lesions  produced  during  disease.  In  many  cases  characteristic — not 
invariably  so — lesions  typical  of  one  infection  may  be  produced  by  a  different 
one — lesions  influenced  by  other  factors  than  species  of  organism— e.g.  age 
of  patient,  route  of  invasion,  secondary  infection,  treatment,  etc. — possibility 
of  excluding  such  factors  by  inoculation.  2.  Variation  in  lesion  caused  by 
artificial  inoculation.  Method  "standardises"  lesion — lesions  said  to  be 
invariable  under  these  conditions.  B.  pseudo-dip ht her iae  distinguished 
from  B.  coli — avian  tubercle  bacillus  distinguished  from  human  type — 
S.  mastitidis  distinguished  from  S.  anginosa  and  S.  pyogenes — pneumo- 
coccus of  lobar  pneumonia  distinguished  from  that  of  lobular  pneumonia. 
Are  the  lesions  cawed  by  artificial  means  invariable? — certainly  very 
constant — e.g.  tubercle  bacillus — but  not  absolutely  fixed — e.g.  a  strain  of 
B.  diph.  causes  lesions  of  rabies — a  strain  of  S.  mastitidis  loses  its  power 
to  cause  typical  lesion — various  types  of  tubercle  bacilli  fail  to  cause  their 
typical  lesions.  Two  strains  causing  different  lesions  arise  from  single  strain 
during  cultivation — D.  lanceolatus  capsulatus  isolated  from  different  organs 
causes  different  lesions — type  of  lesion  altered  if  organism  first  grown 
anaerobically.  Every  aspect  of  pathogenicity  subject  to  variation. 

Other  characters  of  bacteria  equally  variable.  (94 — 106) 


CHAPTER  VIII 

THE  POSSIBLE  OCCURRENCE  OF  TRANSMUTATION 
IN  THE  LIVING  BODY 

Organisms  closely  resembling  each  other  except  in  pathogenicity  often 
found  associated — can  one  of  these  be  a  derivative  of  the  other? — e.g. 
B.  anthracis  and  B.  anthracoides,  in  hides  of  cattle.  Other  instances  in 
the  human  body.  B.  coli  and  B.  typhosus.  Klebs-Loeffler  bacillus  and 
Hofmanris  bacillus—  pathogenesis — fermenting  properties — seasonal  pre- 
valence— during  convalescence  from  diphtheria — recent  work.  Staph. 
epidermidis  and  staph.  pyogenes — pathogenesis — fermenting  properties — 


SYNOPSIS  xv 

pigment  formation.  The  meningococcus  and  M.  catarrhalis — morpho- 
logy— fermenting  properties — pathogenesis — mixed  infection — habitat.  The 
meningococcus  and  the  pneumococcus — symptoms  produced — common  com- 
plications— seasonal  prevalence — age  incidence  and  mortality— distribution. 
Such  transition  less  credible  than  it  appears — but  not  less  credible  than 
instances  known  to  occur— saprophy tic  and  parasitic  types  of  pneumococcus. 
Conclusions.  (107—115) 


CHAPTER  IX 

SUPPOSED  INSTANCES  OF  TRANSMUTATION  BROUGHT 
ABOUT  EXPERIMENTALLY 

I.  MAJOR  HORROCKS'S  EXPERIMENTS  (Journal  of  R.A.M.C.  Vol.  xvi). 
Importance  of  the  claims  made  by  him.    Criticism.    Possibilities  to  be 
considered — purity    of  original   strain,   peritoneum  possibly  not  sterile, 
possible  contamination  from  skin,  possible  invasion  from  gut  before  or  after 
death,  continuity  of  strain  not  confirmed  by  reversion  or  presence  of 
intermediate  forms,  different  results  obtained  on  repeating  experiments, 
results  possibly  explained  by  variation.    Criticism.    Conclusions. 

II.  RELATIONSHIP  BETWEEN  PARATYPHOID  ORGANISMS. 

A.  Schmitfs  experiments.  Experiment  I.  "Flugge"  type  given  to  calf 
in  food,  injected  beneath  skin — second  strain  isolated  from  blood.  Experi- 
ment II.  Second  strain  injected  into  nasopharynx  of  second  calf— third 
strain  isolated  from  blood  and  injected  into  vein,  fourth  strain  recovered 
after  death— later  strains  resembled  B.  Gaertner  in  agglutination.  Possible 
fallacies,  (a)  contamination  in  original  strain  ?  (6)  contamination  in  bodies 
of  calves  ? — not  absolutely  excluded  by  agglutination  tests — B.  Gaertner  in 
intestines  of  healthy  calves,  possible  increase  in  numbers  and  virulence  due 
to  local  inflammation,  might  lead  to  systemic  invasion,  (cf.  saprophytes  in 
inflamed  uterus)  and  fresh  agglutination  reactions  of  blood  serum. 

.  Experiments  of  Miihlens,  Dahm  and  Furst.  Mice  fed  on  infected 
meat— faeces  of  some  contained  B.  Aertryck,  of  others  B.  Gaertner— due 
to  transmutation  ?  Possible  fallacies,  (a)  contamination  of  original  source  ? 
(&)  contamination  in  bodies  of  mice? — control — B.  Aertryck  in  healthy 
mice,  presence  possibly  overlooked  ?— their  appearance  favoured  by  in- 
flammation and  disturbed  function  of  bowel. 

C.  The  Author's  experiments.  Experiment  I.  Guineapigs  given  B. 
Gaertner  in  food — B.  Gaertner  and  B.  Aertryck  isolated  from  faeces  at 
different  times — control — two  organisms  transmutable  ? — intestinal  bacteria 
undetected  if  few  in  number — disturbed  function  of  bowel  reveals  their 
presence — multiply  in  inflamed  intestine  (cf .  B.  coli  in  cholera) — such  factors 
may  explain  result  of  experiment — qualification — proof  that  in  disordered 
intestine  unsuspected  organisms  make  their  appearance.  Experiment  II. 
Faeces  of  six  guineapigs  examined — B.  proteus  found  in  one  case — guinea- 


xvi  SYNOPSIS 

pigs  given  unwholesome  food — faeces  again  examined — B.  proteus  found  in 
four  cases.  Conclusions — suggest  presence  of  secondary  invaders  in  experi- 
ments quoted — possible  error  from  identifying  organism  by  agglutination — 
variable  agglutination  of  paratyphoid  organisms.  Application  to  experiments 
quoted.  Results  no  evidence  of  transmutation.  (116 — 139) 


CHAPTER  X 

SUMMARY 

All  characters  of  bacteria  show  variation — "spontaneous"  or  "impressed" 
— modifying  influences  already  discussed  (Chap.  n).  Variation  may  be  ap- 
parent only.  Apparently  spontaneous  variation  may  be  due  to  unrecognised 
influences.  Variation  itself  may  be  specific  (morphology  of  B.  diphtheriae 
and  S.  scarlatinae).  No  one  character  specific — variation  need  not  imply 
loss  of  specific  character  (morphology  of  B.  coli}.  Analogy  of  regiment  of 
soldiers  and  crowd  of  pitmen.  Variation  may  be  specific  because  it  indicates 
racial  character.  Many  variations  represent  past  stages  in  evolution  (Mor- 
phology, Chap,  iv) — others  represent  new  steps  in  evolution  (Fermenting 
Power  and  Virulence,  Chaps,  v  and  vi). 

TRANSMUTATION  DIFFERS  FROM  VARIATION  IN  DEGREE  ONLY—  different 
species  derived  from  a  common  stock — differentiation  more,  advanced  in 
some  than  others — reversion  therefore  differently  interpreted — necessity  of 
regarding  characters  as  a  whole  and  their  stability — danger  of  relying  upon 
one  character  alone  already  shown  (Chap,  iv-vn).  Analogy  of  human  race 
groups.  Variation  may  indicate  recent  environment — and  so  reveal  source 
of  particular  strain  (streptococcus  from  milk — general  coli  infection  from 
biliary  passages). 

STABILITY  OF  VARIATIONS.  "Spontaneous"  variations.  (1)  Imperfect 
development — tend  to  disappear.  (2)  Senility  or  lowered  vitality — tend  to 
persist.  (3)  Atavistic  tendencies — tend  to  recur.  (4)  Fresh  stage  in  evolu- 
tion— therefore  unstable.  Two  variations  constantly  associated — both  due 
to  lowered  vitality — both  due  to  higher  evolution — both  due  to  imperfect 
development  or  degeneracy.  "Impressed"  variations — may  lapse  when  in- 
fluence withdrawn — may  persist  for  a  time — may  appear  permanent — danger 
of  assuming  variation  is  permanent — examples — ability  to  ferment  sugar  or 
produce  pigment — inability  to  ferment  sugar  or  display  virulence.  Duration 
of  impressed  variation.  (1)  If  only  part  of  strain  varies  it  may  appear  to 
revert— danger  of  assuming  reversion  has  occurred.  (2)  If  readily  acquired 
is  long  retained— if  slowly  acquired  quickly  lost  (ability  of  B.  typhosus  to 
ferment)— not  true  of  spontaneous  variations.  (3)  The  longer  the  training 
the  more  lasting  the  effect  (streptococcus  at  different  stages  in  disease- 
ability  of  B.  typhosus  to  ferment).  Same  principle  governs  development  of 
races.  Absence  of  reversion  does  not  imply  inability  to  revert  (pigment 


SYNOPSIS  xvii 

production  by  B.  ruber— mycelial  development  of  B.  diph.}.  Tendency  to 
revert  does  not  imply  loss  of  specific  character.  Variation  differs  from 
transmutation  in  degree  alone. 

TRANSMUTATION  DIFFERS  FROM  EVOLUTION  IN  DEGREE  ALONE.  Analogy  of 
different  branches  of  family.  Possibility  of  transmutation.  Saprophytic  and 
parasitic  pneumococcus.  Other  examples  already  discussed  (Chap.  vm). 
Experiments  suggesting  transmutation  already  discussed  (Chap.  ix).  1st 
series,  strains  not  guaranteed  pure,  results  explained  by  variation.  2nd 
series,  results  explained  by  variation,  secondary  invasion  not  excluded. 
Transmutation  improbable.  Enzyme  theory  of  disease.  (140 — 152) 


CHAPTER  XI 

THE  ENZYME  THEORY  OF  DISEASE 

Predicates  disease  not  due  to  bacteria  but  to  their  ferments.  (1)  Ac- 
quisition and  loss  of  pathogenic  powers.  (2)  Different  organisms  may  cause 
same  type  of  disease — rabies  due  to  B.  diph.  (3)  Same  organism  causes 
different  types  of  disease — in  different  epidemics  (B.  influenzae) — cages 
differ  in  same  epidemic — scarlet  fever  and  puerperal  fever — M.  catarrhalis 
infection  simulating  other  diseases,  coryza,  influenza,  scarlet  fever,  diphtheria, 
typhoid  fever,  cerebrospinal  fever.  (4)  Same  conditions  influence  virulence 
and  fermenting  power,  (a)  antiseptics,  (b)  oxygen— virulence  of  cholera, 
toxicity  of  B.  diph.,  fermenting  power  of  B.  dysent.,  of  streptococcus, 
(c)  temperature — optimum  temperature — digestive  enzyme  in  cold  blooded 
animals — germ  barley — marine  enzymes — fermenting  power  of  B.  coli — 
virulence  of  B.  diph.,  B.  tetani,  B.  anthracis,  etc. — enzymes  killed  at  60°  C. 
and  virulence  destroyed,  (d)  sunlight,  (e)  symbiosis — tetanus  and  pyogenic 
cocci,  B.  coli  and  B.  dentrificans.  (5)  Virulence  due  to  "passage"  through 
an  animal  and  fermenting  power  due  to  growth  in  a  sugar,  (a)  specific, 
(b)  repeated  inoculations  or  subcultures  more  effective,  (c)  power  readily 
acquired  is  easily  maintained,  (rf)  if  recently  lost  is  quickly  regained. 
(6)  Intra-  and  extra-cellular  toxins— intra-  and  extra-cellular  enzymes,  yeast, 
digestive  enzymes — emulsion  of  gland  or  bacteria  more  potent.  (7)  Virulence 
associated  with  fermenting  properties— M.  catarrhalis,  gonococcus  and 
meningococcus,  Hofmann's  bacillus  and  Klebs-Loeffler  bacillus,  B.  coli— both 
due  to  adaptation?  (8)  Living  tissues  defended  by  enzymes.  (9)  Other 
functions  of  bacteria  due  to  enzymes— influenced  by  same  conditions  as 
virulence,  e.g.  pigment  formation.  (10)  These  ferments  separable  from 
bacteria — enzyme  which  liquefies  gelatin  survives  bacteria — passes  filter — 
soluble.  (11)  M.  ureae— enzyme  separable.  (12)  Isolation  of  pathogenic 
enzymes  (pneumococcus).  (13)  Bacteria  deprived  of  a  pathogenic  function 
by  environment — same  conditions  influence  ferment  activity,  (a)  ultra  violet 
rays— pathogenesis  of  B.  anthracis,  (b)  oxygen— power  of  pneumococcus  to 


xviii  SYNOPSIS 

produce  skin  lesion,  (c)  growth  in  milk— fermentation  by  B.  coli,  rash  in 
scarlet  fever,  (d)  effect  of  substances  added  to  media  and  of  drugs  in  disease 
— sod.  benzoate  and  B.  coli— sod.  salicylate  and  acute  rheumatism. 
(14)  Different  symptoms  due  to  different  enzymes? — analogy  with  sugar 
ferments  of  bacteria — complexity  of  action — association  with  particular 
vegetable  and  bacterial  cells — possibility  of  complete  dissociation  of  patho- 
genic enzymes?  (15)  Two  results  obtainable — organisms  deprived  of  patho- 
genic functions  (B.  typhosus) — functions  maintained  in  absence  of  organism, 
e.g.  filter  passers.  (16)  No  enzyme  isolated  which  forms  toxins  outside  the 
body — true  also  of  other  recognised  ferments — artificial  media  differ  from 
vital  fluids.  (17)  The  enzyme  theory  and  transmutation — suggests  transfer 
of  function  possible — certain  conditions  essential.  Analogy  of  ships  at  sea. 
Conclusion.  (153—169) 

CHAPTER  XII 

CONCLUSIONS  (170) 

APPENDIX 

REFERENCES  (171—179) 


INTRODUCTION 

THE  mediaeval  alchemists  conceived  the  idea  of  the  trans- 
mutation of  metals  and  dreamt  of  changing  the  baser  metals 
into  gold.  The  task  which  baffled  them  the  scientists  of  our  own 
generation  seem  destined  to  achieve.  The  transmutation  of 
bacteria  is  a  problem  of  more  recent  date  but  it  bears  a  certain 
resemblance.  If  silver  and  gold  are  the  currency  of  wealth 
by  means  of  which  it  changes  hands,  bacteria  represent  the 
currency  of  disease  by  means  of  which  this  also  is  passed  from 
one  person  to  another.  The  resemblance,  however,  goes  much 
deeper  than  this,  for  just  as  the  metals  have  hitherto  been 
regarded  as  "elements"  of  matter  so  the  functions  of  the 
unicellular  organism  have  been  thought  to  represent  the 
"elements"  of  life.  The  physicist  has  learnt  that  the  so-called 
"elements"  of  matter  are  themselves  composed  of  infinitely 
small  particles  or  "ions" ;  the  pathologist  is  learning  that  the 
functions  of  bacteria  in  many  cases  result  from  the  activity  of 
ultra-microscopic  bodies,  of  the  nature  of  "enzymes."  The 
occurrence  of  transmutation  in  the  case  of  bacteria  would 
prove  as  revolutionary  in  our  conception  of  disease  as  its 
occurrence  in  the  case  of  certain  rare  metals  is  already  proving 
in  our  conception  of  matter. 

The  idea  of  the  permanence  of  characters  in  the  animal 
world  is  at  least  as  old  as  the  question  "Can  the  Ethiopian 
change  his  skin  or  the  leopard  his  spots?"  but  it  is  only  in 
recent  times  that  the  fixity  of  animal  species  has  been 
scientifically  demonstrated. 

Amongst  the  less  highly  organised  structures  of  plant  life 
variation  is  of  more  frequent  occurrence  and,  though  it  is 
not  possible  to  "gather  figs  from  thistles,"  it  is  generally 
acknowledged  that  "species"  in  the  case  of  plants  are  less 
rigidly  defined  than  in  the  animal  world. 

In  the  realm  of  bacteriology  still  simpler  forms  are  met 
with  in  which  are  recognised  the  beginnings  of  both  animal 

D.  1 


2  INTRODUCTION 

and  vegetable  life,  and  amongst  these  variation  is  of  still 
greater  frequency.  This  fact,  confirmed  by  personal  observa- 
tion and  by  a  perusal  of  the  literature  of  the  subject,  suggested 
to  the  writer  the  question  whether  actual  transmutation  of 
species  might  not  occur  amongst  bacteria,  and  it  was  in  the 
hope  of  answering  this  question  that  the  investigation  here 
recorded  was  undertaken. 

An  endeavour  was  made  in  the  first  instance  to  collect 
the  published  records  of  all  experiments  in  which  transmuta- 
tion was  alleged  to  have  occurred.  These  were  found  to  be 
few  in  number.  In  the  second  place,  a  series  of  experiments 
was  carried  out  by  the  writer  with  the  object  of  disproving 
the  contention  put  forward  in  one  of  these  cases.  Thirdly, 
with  a  view  to  criticising  the  claim  made  in  the  remaining 
cases,  and  in  the  hope  that  it  might  throw  some  light  on  the 
problem  of  transmutation  as  a  whole,  a  study  of  the  subject 
of  variation  amongst  bacteria  was  undertaken.  The  material 
on  which  it  is  based  has  been  collected  from  the  scattered 
literature  of  the  subject.  With  a  few  exceptions,  only  papers 
written  in  English  have  been  consulted. 

In  Chapter  I  the  scope  of  the  enquiry  is  outlined.  In 
Chapter  II  the  conditions  which  modify  the  characters  of 
bacteria  are  enumerated  and  in  Chapter  III  the  value  of  the 
evidence  adduced  in  proof  of  such  modification  having 
occurred  is  considered.  Examples  of  variation  are  then  studied 
in  detail  and  their  significance  is  discussed,  reference  being 
made  more  particularly  to  morphological  characters,  fer- 
menting properties,  virulence,  and  pathogenesis  (Chapters 
IV — VII).  In  Chapter  VIII  the  possibility  of  transmutation 
occurring  in  the  animal  body  is  considered.  In  Chapter  IX 
instances  of  supposed  transmutation  are  examined.  In  Chapter 
X  the  subject  is  reviewed  at  length  and  the  results  of  the 
investigation  summarised.  In  Chapter  XI  the  Enzyme  theory 
of  disease  is  discussed,  together  with  its  bearing  upon  the 
subject  of  transmutation.  In  Chapter  XII  the  author's  conclu- 
sions are  briefly  stated.  References  are  given  in  the  Appendix. 


CHAPTER  I 

THE  SCOPE  OF  THE  ENQUIRY 

DEFINITION  OF  TERMS 

THE  phrase  "transmutation  of  bacteria"  is  not  synonymous 
with  "evolution  of  bacteria."  "Evolution"  is  the  gradual  de- 
velopment of  new  species  and  tends  towards  further  differentia- 
tion. "Transmutation"  is  the  changing  of  members  of  one 
recognised  species  into  those  of  another  and,  if  proved,  would 
tend  towards  unification  by  undermining  existing  barriers. 

There  is  no  reason  to  doubt  and  abundant  evidence  to 
support  the  opinion  that  in  this  field  of  life,  as  in  others,  the 
forces  of  natural  selection  and  the  survival  of  the  fittest  have 
been  at  work  and  have  resulted,  in  the  course  of  ages,  in  the 
evolution  and  differentiation  of  the  various  types  of  bacteria 
which  we  recognise  and  distinguish  today. 

Andre wes,  in  the  Horace  Dobell  Lecture  for  1906,  "traced 
the  evolution  of  streptococci  from  the  condition  of  harmless 
mineral-feeders,  through  that  of  saprophytism  in  the  alimen- 
tary canal,  to  the  development  of  weak  powers  of  parasitism 
which  have  culminated,  in  certain  instances,  in  the  fully 
developed  property  of  aggressive  parasitism  seen  in  the 
streptococcus  pyogenes." 

He  showed  how,  at  different  stages,  natural  selection  and 
the  survival  of  those  best  adapted  to  the  environment  in  which 
they  found  themselves,  resulted  in  the  permanent  acquisition 
of  new  characters,  such  as  the  ability,  when  they  had  once 
entered  upon  a  saprophy tic  career  in  the  alimentary  canal,  to 
flourish  most  vigorously  at  the  body  temperature  of  their  host 
and  to  utilise  the  foodstuffs  available  in  their  new  habitat ;  to 
resist  desiccation  during  the  intervals  between  their  discharge 
from  one  host  and  their  reception  by  another ;  and  later  still 
to  support  themselves  in  the  actual  living  tissues  of  the  host 

1—2 


4  THE  SCOPE  OF  THE  ENQUIRY  [CH.  i 

and  to  defend  their  position  there  by  the  manufacture  of 
haemolysins  and  toxins. 

This  process  of  evolution  is,  no  doubt,  going  on  continually 
in  bacteria  as  in  higher  forms  of  life.  It  is  rendered  possible 
in  both  cases  by  the  occurrence  of  natural  variation.  This 
variation  in  bacteria  is  of  two  kinds,  namely,  spontaneous  or 
intrinsic  variation  between  the  individuals  of  a  pure  culture — 
that  is  to  say,  bacteria  derived  from  a  single  organism, — and 
impressed  variation,  the  effect  of  special  environmental  con- 
ditions upon  a  succession  of  bacterial  generations,  due  either 
to  the  direct  reaction  of  the  bacterial  protoplasm  to  the 
environment,  or  to  selection  acting  upon  slight  spontaneous 
variations  and  producing  a  cumulative  effect. 

It  is  reasonable  to  expect  that  amongst  the  bacteria  natural 
variation  would  occur  with  greater  frequency  than  amongst 
higher  forms  of  life  for,  being  unicellular  organisms,  changes 
in  their  environment  can  operate  directly  upon  the  germ  plasm. 
Moreover  the  common  method  by  which  bacteria  multiply, 
namely  the  division  of  the  parent  cell  into  two  daughter  cells, 
ensures  the  ready  transmission  of  any  acquired  character  from 
parent  to  offspring.  The  variation  in  character  may  be  said  to 
be  retained  by  the  daughter  cells  rather  than  transmitted  to 
them.  Such  retention  of  parental  characters  by  the  daughter 
cells  is  not,  however,  invariable.  For  example,  McDonald 
(1908)  has  published  photographs  of  a  young  culture  of  the 
meningococcus  showing  diplococci  in  which  one  member  is 
stained  while  the  other  is  not.  Thirdly,  such  variations  would 
be  more  readily  noted  in  their  case  since  as  many  as  30  or 
40  successive  generations  may  be  observed  in  the  course  of 
24  hours.  In  the  case  of  some  bacteria  division  may  occur  as 
frequently  as  once  every  17  minutes  (Barber,  1908).  Yet  a 
fourth  factor  might  be  mentioned,  namely  the  ease  with  which 
the  environment  of  any  strain  of  organisms  can  be  modified  in 
any  direction  and  to  any  extent. 

As  a  matter  of  fact  examples  of  such  variation,  as  we  shall 
show,  are  innumerable,  no  matter  what  particular  property  or 
character  of  bacteria  we  investigate.  Differences  occur  in  size 
and  shape,  in  staining  properties,  in  power  of  growth  on  various 


CH.  i]  THE  SCOPE  OF  THE  ENQUIRY  5 

media,  in  viability,  in  virulence,  in  the  power  to  ferment  sugars, 
and  so  on. 

The  environment  in  which  bacteria  grow  and  multiply 
tends,  in  the  course  of  time,  to  "fix"  some  of  these  variations 
by  offering  to  the  possessors  of  them  a  better  chance  of  survival 
or  perpetuation,  so  that  they  ultimately  become  characteristic 
of  a  new  species.  This  is  evolution  through  natural  selection. 

By  a  similar  process  of  artificial  selection,  as  will  be  shown, 
we  can  encourage  variation  in  almost  any  direction  we  choose. 
"Within  certain  limits  the  simple  forms  of  life  are  able  to  adapt 
themselves  to  their  surroundings  and  the  adaptation  cannot 
be  ascribed  to  chance  for,  with  a  given  environment,  the  one 
particular  alteration  in  properties  surely  results."  (Adami, 
1910.)  If  we  so  vary  the  characters  of  a  member  of  one  species 
that  it  comes  ultimately  to  possess  all  the  characters  of  a 
member  of  another  species,  that  is  "transmutation."  The 
question  is,  how  far  can  we  go  in  this  direction,  and  to  what 
extent  are  the  recognised  species  of  bacteria  really  fixed  in 
their  characters? 

THE  MEANING  OF  SPECIES. 

The  objection  may  be  raised  at  this  point,  that  the  phrase 
"transmutation  of  species"  involves  a  contradiction  in  terms, 
since  the  very  definition  of  "species"  excludes  the  possibility 
of  transmutation.  This  leads  us  to  a  further  question,  namely, 
what  do  we  mean  by  the  word  "species"  as  applied  to 
bacteria? — in  other  words,  what  determines  our  present 
classification  ? 

The  distinction  between  different  species  of  bacteria  and 
their  recognition  depends  upon  the  observation  of  their  charac- 
ters— morphological,  biological,  chemical,  physiological  and 
pathological.  Briefly  enumerated  these  are  as  follows : — the 
naked  eye  appearance  of  colonies  and  of  a  stab  culture: 
microscopic  appearances,  size,  shape,  motility,  adhesiveness: 
method  of  generation  and  life  history,  involution  forms :  power 
to  produce  pigment :  staining  properties :  cultural  characters, 
power  of  growing  on  different  media,  in  the  presence  or  absence 
of  oxygen,  and  under  different  conditions  of  temperature  and 


6  THE  SCOPE  OF  THE  ENQUIRY  [CH.  i 

moisture,  with  or  without  production  of  gas  or  odour ;  power 
to  liquefy  gelatin,  to  reduce  neutral  red,  to  clot  milk,  to  ferment 
various  carbohydrate  substances :  power  to  form  agglutinins 
and  susceptibility  to  agglutination :  viability  under  different 
conditions :  virulence  or  the  nature  of  the  toxins  they  produce : 
pathogenicity  or  the  nature  of  the  lesions  they  cause  and  the 
kind  of  animal  susceptible  to  their  invasion. 

It  is  seen  from  this  list  that  the  characteristic  qualities  of 
bacteria  are  very  numerous  and  it  would  be  thought  that  their 
classification  would  on  this  account  be  very  thorough  and 
complete.  But,  as  will  be  shown  in  the  course  of  this  enquiry, 
every  one  of  these  characters  is  liable  to  variation  and  the 
occurrence  of  these  variations  renders  the  task  of  classification 
very  difficult  and  in  many  cases  uncertain. 

If  certain  characteristics  were  invariable,  even  though 
others  varied,  a  definite  criterion  would  be  afforded,  but  where 
all  alike  are  subject  to  modification  the  division  into  species 
is  necessarily  an  arbitrary  one.  Amongst  the  higher  animals, 
where  sexual  production  prevails,  mutual  fertility  or  sterility 
offers  a  guide  in  determining  the  limits  of  species.  Here  no 
hard  and  fast  line  can  be  drawn.  Nevertheless  we  see  exhibited 
amongst  bacteria,  in  the  words  of  De  Bary,  "  the  same  periodi- 
cally repeated  course  of  development  within  certain  empirically 
determined  limits  of  variation,"  which  is  considered  to  justify 
the  recognition  of  a  species. 

Many  species  of  bacteria  do  show  characters  apparently 
quite  fixed  and  rigid.  The  anthrax  bacillus  and  the  tetanus 
bacillus  are  quite  as  good  species  in  the  natural  history  sense 
as  any  that  can  be  found  amongst  flowering  plants.  But  the 
classification  of  others  is  still  a  matter  of  dispute.  This  can 
be  illustrated  by  reference  to  the  streptococci. 

THE  CLASSIFICATION  OF  THE  STREPTOCOCCI. 

Marmorek  held  the  opinion  that  the  human  streptococci 
constituted  one  species.  "He  based  his  chief  argument  on  the 
observation  that  bouillon  in  which  one  sort  of  streptococcus 
had  grown  would  not  serve  afterwards  as  a  culture  medium 
for  any  other  streptococcus,  and  that  the  same  haemolytic 


CH.  i]  THE  SCOPE  OF  THE  ENQUIRY  7 

power  was  possessed  by  them  all."  (Andrewes  and  Border, 
1906.) 

In  1891  von  Lingelsheim  (ibid.)  proposed  a  division  into  two 
groups  according  to  the  length  of  chains  formed:  "strepto- 
coccus brevis"  and  "streptococcus  longus."  Andrewes  and 
Horder  (1906)  with  a  view  to  further  classification  on  the  same 
lines  suggested  the  adoption  of  the  terms  brevissimus,  brevis, 
medius,  longus,  longissimus,  conglomeratus.  The  quality  of 
cohesiveness  by  itself  was,  however,  considered  too  trivial  a 
character  to  base  a  fundamental  classification  upon. 

The  power  of  retaining  stains  was  found  to  offer  no  means 
of  differentiation,  since  all  stained  well. 

Minute  differences  in  their  mode  of  growth  on  different 
media  were  found  to  be  too  inconstant  to  be  of  any  value, 
though  Schottmuller  (quoted  by  Muir  and  Ritchie)  attempted  to 
classify  the  streptococci  according  to  the  appearance  of  colonies. 

Classification  according  to  pathogenicity  and  virulence 
appeared  to  have  the  advantage  of  being  practical  and  signifi- 
cant from  a  clinical  standpoint.  Virulence,  however,  was 
likewise  found  to  be  an  inconstant  character,  being  lost  and 
regained  with  great  readiness  by  these  organisms.  It  was  lost 
after  a  few  days  on  certain  culture  media.  On  the  other  hand, 
after  a  few  "passages"  through  a  susceptible  animal,  a  strepto- 
coccus of  feeble  virulence  might  become  intensely  pathogenic. 
Clinical  experience  confirms  this  variability  in  virulence.  "  One 
and  the  same  strain  of  streptococcus  may  at  different  stages 
in  its  career  produce  now  a  rapidly  fatal  septicaemia,  now  a 
spreading  erysipelas,  now  a  localised  suppuration  and  now  no 
effect  at  all"  (Andrewes  and  Horder,  1906),  so  that  the  degree 
of  virulence  was  an  uncertain  aid  to  classification.  The 
streptococcus  erysipelatis,  for  instance,  is  no  longer  considered 
on  account  of  its  virulence  to  be  a  distinct  species  from  the 
streptococcus  pyogenes. 

Agglutination  tests  have  not  been  found  to  be  sufficiently 
specific. 

Marmorek's  contention,  therefore,  for  the  unity  of  species  of 
the  human  streptococci  continued  to  hold  the  field  successfully 
until  the  introduction  of  Gordon's  tests. 


8  THE  SCOPE  OF  THE  ENQUIRY  [OH.  i 

Gordon  (1903-4)  isolated  from  human  saliva  300  strains  of 
streptococci.  He  tested  separately  the  power  of  these  different 
strains  to  ferment  various  substances,  consisting  of  14  carbo- 
hydrates, 13  glucosides,  and  6  polyatomic  alcohols.  Many  of 
these  test  substances  proved  of  no  differential  value  as  regards 
streptococci,  either  because  they  were  uniformly  attacked  by 
all  or  because  no  streptococci  could  ferment  them,  but  he  was 
led  to  select  a  series  of  seven  substances — namely  saccharose, 
raffinose,  inulin,  salicin,  coniferin  and  mannite — as  being  of 
special  value  as  tests  for  streptococci,  and  to  these  he  added 
two  further  tests — the  clotting  of  milk  and  the  reduction  of 
neutral  red  under  anaerobic  conditions.  By  such  means  he 
was  enabled  to  distinguish  48  chemical  varieties. 

Houston  (1903-4)  applied  the  same  tests  (with  the  omission 
of  one — the  action  on  coniferin)  to  300  strains  of  streptococci 
derived  from  human  faeces  and  was  able  to  distinguish 
40  chemical  varieties  amongst  them. 

Gordon  demonstrated  that  these  chemically  different  strains 
were  remarkably  constant  in  their  reactions  and  this  was 
confirmed  later  by  the  work  of  Andre wes  and  Horder  (1906), 
who  tested  his  strains  and,  in  addition,  some  200  new  strains 
derived  from  foci  of  disease  in  human  beings.  "  Gordon  himself 
was  careful  to  abstain  from  claiming  specific  value  for  his 
different  chemical  types  and  he  did  not  venture  to  propose  any 
reasoned  scheme  of  scientific  classification  based  upon  his 
tests."  Andrewes  and  Horder  attempted  to  do  this.  They 
collected  from  various  sources  particulars  of  the  behaviour  of 
over  1200  different  strains  of  streptococci  when  subjected  to 
Gordon's  tests.  As  a  result  they  found  that  these  1200  strains 
fell  into  some  half  a  dozen  main  groups.  By  adding  one 
further  test — the  power  of  growth  in  gelatin  at  20°  C. — and  by 
taking  into  consideration  also  the  morphological  characters 
and  pathogenesis,  they  were  able  to  define  five  varieties  of 
streptococci  which  they  regarded  as  of  "approximately  specific 
value"  though  connected  by  a  multiplicity  of  intermediate 
varieties.  They  named  these  S.  anginosa,  S.  salivarius, 
S.  faecalis,  S.  pyogenes  and  8.  pneumococcus. 

Though  confirming,  on  the  whole,  the  stability  of  the 


CH.  i]  THE  SCOPE  OF  THE  ENQUIRY  9 

reactions  constituting  the  tests,  these  observers  noticed  that 
variation  in  virulence  was  sometimes  accompanied  by  changes 
in  chemical  behaviour.  They  also  acknowledged  that  slight 
differences  in  the  composition  of  the  media  might  possibly 
affect  the  series  of  reactions  to  some  slight  extent. 

Subsequently  Ainley  Walker  (1911)  offered  evidence  to 
show  that  greater  differences  in  the  media  do  actually  affect 
the  reactions  to  a  remarkable  extent,  so  much  so  as,  in  his 
opinion,  to  invalidate  any  claim  that  they  should  be  re- 
garded as  specific,  and  he  fell  back  on  the  position  held  by 
Marmorek. 

Still  later  Jensen  and  Holth,  after  a  prolonged  investiga- 
tion, came  to  the  opposite  conclusion,  that  is  to  say  in  favour 
of  the  stability  of  the  differences  brought  out  by  Gordon's 
tests,  but  they  also  showed  that  these  differences  were  in  no 
way  closely  related  to  virulence  or  pathogenic  action  so  that 
the  method  of  classification  founded  on  them  was  imperfect. 

We  have  thus  demonstrated  that  the  term  "species"  when 
applied  to  bacteria  must  be  interpreted  much  more  loosely 
than  in  the  case  of  plants  or  the  higher  animals  and  the  phrase 
"transmutation  of  species"  is  thereby  absolved  of  the  accusa- 
tion of  being  self- contradictory. 

The  aim  of  this  paper  is  to  show  how  far  transmutation 
does  occur,  and  what  is  its  significance. 

It  will  be  obvious,  from  what  has  already  been  said,  that 
in  considering  the  evidence  of  transmutation  it  will  be  neces- 
sary to  consider  also  the  evidence  of  variation.  The  difference 
between  the  two  is  one  of  degree  only.  A  member  of  one 
"species"  of  bacteria  is  distinguished  from  a  member  of 
another  "  species  "  by  its  morphological  and  other  characters. 
If  these  characters  become  altered,  within  certain  limits,  the 
process  may  be  regarded  simply  as  variation ;  if  outside  these 
limits  it  must  be  regarded  as  transmutation. 

We  have  first  of  all  to  consider  then,  what  are  the  possi- 
bilities in  the  direction  of  such  alteration  in  character. 


10  THE  SCOPE  OF  THE  ENQUIRY  [CH.  i 

A  CONSIDERATION  OF  THE  POSSIBILITIES. 

The  various  possibilities  to  be  considered  are  five  in 
number.  It  will  be  seen  that  the  first  three  are  instances  of 
variation  and  the  remaining  two,  instances  of  transmutation. 

1.  Simple  variation.    Modifications  may  occur  in  the 
characters  of  an  organism,  involving  either  the  loss  of  some 
feature  previously  regarded  as  characteristic  or  the  acquisition 
of  some  other  feature  not  hitherto  considered  to  be  so, — such 
modifications  not  being  so  numerous,  however,  or  so  funda- 
mental, as  to  lead  to  any  doubt  as  to  the  proper  identification 
of  the  organism.   For  example,  Twort  (1907)  succeeded,  in  the 
course  of  two  years,  in  training  a  strain  of  B.  typJiosus  to 
ferment  lactose,  and  both  Twort  (1907)  and  Penfold  (1910  A) 
produced  pure  strains  capable  of  fermenting  dulcite,  which 
the  usual  variety  of  typhoid  bacillus  is  practically  unable  to 
do.    These  new  strains  retained  qualitatively  all  the  other 
properties  of  the  B.  typhosus  unchanged.     Similarly  Miss 
Peckham  (1897)  induced  indol  formation  in  numerous  strains 
of  B.  typhosus. 

2.  Variations  in  different  directions  associated.    The 
acquirement  of  some  fresh  character  may  be  associated  with 
the  loss  simultaneously  of  some  other  character  previously 
possessed  or,  with  the  acquisition  of  a  second  new  character, 
and  in  some  cases  this  association  may  prove  to  be  invariable 
under  the  same  modifying  conditions  (vide  p.  146).    For 
example,  Eyre  and  Washbouru  (1899)  observed  that  a  non- 
virulent  strain  of  the  pneumococcus  growing  readily  at  20°  C. 
could  by  "passage"  be  converted  into  a  highly  virulent  strain 
which  was  then  unable  to  grow  at  a  temperature  below  37°  C. 
The  reverse  change  showed  the  same  relation  between  the 
virulence  of  the  organism  and  the  temperature  at  which  it 
would  grow. 

Jenner  (1898)  was  able  to  revert  B.  coli  capsulatiis  to  an 
unencapsulated  form  by  cultural  methods  and  found  that  the 
new  variety  had  lost  the  power  to  coagulate  milk,  and  instead 
of  being  highly  pathogenic  to  white  mice  had  become  much 
less  so  or  even  non-pathogenic. 


CH.  i]  THE  SCOPE  OF  THE  ENQUIRY  11 

The  acquirement  of  power  to  ferment  a  certain  carbo- 
hydrate may  coincide  with  the  loss  of  fermenting  power  in 
other  directions. 

Penfold  (1910-11)  has  shown  that  the  development  of  new 
fermenting  powers  on  the  part  of  B.  typhosus  towards  lactose 
and  dulcite  is  frequently  associated  with  the  formation  of 
papillae  on  its  colonies.  Diminished  gas-production  in  glucose 
media  on  the  part  of  certain  coliform  organisms  (B.  Grunthal, 
etc.)  was  likewise  associated  with  papillae  formation. 

The  same  observer  found  that  colonies  of  B.  typhosus  which 
had  lost  the  property  of  fermenting  glycerine  showed  impaired 
agglutinability  also,  though  typical  fermenting  colonies  on 
the  same  plate  were  normal  as  regards  agglutination. 

Adami,  Abbott  and  Nicholson  (1899)  found  that  the  as- 
sumption of  coccic  and  diplococcic  forms  by  B.  coli  in  the 
organs  of  healthy  animals  was  associated  with  a  loss  of  power 
to  ferment  carbohydrates  and  to  produce  indol. 

Gordon  (1900-1)  observed  that  the  tendency  of  the  strep- 
tococcus of  scarlet  fever  to  assume  a  bacillary  form  was 
abolished  by  "passage"  and  at  the  same  time  its  virulence 
was  increased. 

Rosenow  (1914)  obtained  a  strain  of  streptococci  from  the 
throat  in  a  case  of  scarlet  fever,  which  yielded  on  blood  agar 
two  distinct  kinds  of  colonies.  These  displayed  marked  differ- 
ences in  their  fermenting  power  and  also  in  their  pathogenicity. 

Many  other  instances  might  be  given. 

3.  The  development  of  intermediate  forms,  i.e.  the  possible 
derivation  from  one  or  other  of  two  known  species  of  forms 
intermediate  between  them  in  their  characters.  For  example, 
W.  J.  Wilson  obtained  from  the  urine  of  a  supposed  typhoid 
carrier  (1910),  and  also  from  the  urine  in  certain  cases  of 
cystitis  and  pyelitis  (1908),  coliform  organisms  intermediate 
in  their  characters  between  B.  typhosus  and  B.  coli  communis 
and  derived  presumably  from  B.  typhosus  in  one  case  and 
from  B.  coli  in  the  others.  Many  other  observers  have 
described  organisms  resembling  both  B.  typhosus  and  B.  coli 
communis  in  their  characters.  Klotz  (1906)  has  described 
such  an  organism,  isolated  from  water,  and  called  by  him 
Bacillus perturbans.  Mcnaught  (1905)  described  two  varieties, 


12  THE  SCOPE  OF  THE  ENQUIRY  [CH.  i 

also  derived  from  water,  under  the  term  Bacillus  typhosm 
simulans. 

One  organism,  for  example,  described  by  Wilson  (1910), 
resembled  B,  typhosus  in  forming  acid  without  gas  in  glucose 
and  in  failing  to  ferment  lactose  at  37°  C. ;  but  it  failed  to 
agglutinate  with  typhoid  serum,  and  it  resembled  B.  coli  in 
producing  acid  and  much  gas  from  mannite  and  in  fermenting 
lactose  at  22°  C. 

The  Bacillus  perturbans  of  Klotz  was  agglutinated  by 
high  dilutions  of  typhoid  serum  (1-1550  in  15  minutes)  and 
it  produced  slight  acidity  in  milk  without  coagulation ;  but  it 
differed  from  B.  typhosus  in  fermenting  both  lactose  and 
saccharose,  in  giving  the  neutral  red  reaction  and  forming 
indol,  and  in  other  ways. 

Major  Hor rocks  obtained  from  the  urine  of  a  patient 
convalescent  from  typhoid  fever  a  typical  strain  of  B.  typhosus 
which  however  on  subculture  gave  rise  to  an  organism  inter- 
mediate in  its  characters  between  B.  typhosus  and  B.  coli 
(vide  p.  118). 

4.  Slight  changes  in  closely  allied  organisms,  i.e.  the 
possibility  in  the  case  of  closely  allied  organisms  of  a  modifica- 
tion in  the  few  distinguishing  features  they  possess,  so  that 
they  may  appear  to  change,  the  one  into  the  other.    For 
example  Schmitt  (1911)  concluded  from  his  experiments  that 
paratyphoid  bacilli  of  the  'Fliigge'  type  and  of  the  'Gaertner' 
type,  generally  regarded  as  distinct  species,  could  be  trans- 
formed from  one  into  the  other  in  the  animal  body. 

5.  A  complete  change  in  characters,  i.e.  the  possibility  of 
the  occurrence,  more  or  less  suddenly,  of  a  complete  change 
simultaneously  of  all  the  characters  of  an  organism,  or  at  least 
of  all  the  fundamental  ones  by  which  it  was  distinguished. 
For  example,  Major  Horrocks  (1911)  concluded  that  he  had 
been  able  gradually  to  modify  a  strain  of  B.  typhosus,  by 
changes  in  its  environment,  to  such  an  extent  that  it  assumed 
eventually  the  characteristics  of  a  Gram-positive  coccus  having 
the  cultural  characters  of  Streptococcus  faecalis. 

Before  these  several  possibilities  are  studied  more  fully, 
the  conditions  which  modify  the  characters  of  bacteria  will  be 
mentioned  and  examples  given  under  each  head. 


CHAPTER  II 

CONDITIONS  MODIFYING  THE  CHARACTERS 
OF  BACTERIA 

THE  factors  which  appear  to  influence  the  growth  and  de- 
velopment of  bacteria  and  to  produce  modification  in  their 
characters  are  many  in  number  and  diverse  in  nature,  and  it 
is  often  impossible  to  state  with  certainty  which  of  these 
various  factors  is  the  one  primarily  responsible  for  the 
modification  observed  in  a  particular  case. 

1.  Many  variations  appear  to  be  spontaneous,  not  due, 
that  is  to  say,  to  any  external  agency,  but  the  result  of 
developmental  or  atavistic  tendencies  inherent  in  the  organism 
itself.   We  know  as  little  of  the  nature  of  these  tendencies  to 
variation  as  we  know  of  the  nature  of  those  which  control 
normal  development  and,  in  the  vast  majority  of  cases,  prevent 
variation  occurring. 

Some  spontaneous  variations  are  examples  of  "pleomorph- 
ism"  and  represent  stages  in  the  life  history  of  the  individual 
organism  or  of  the  race.  Others  cannot  be  explained  in  this 
way.  For  example,  one  component  of  a  diplococcus  may  retain 
a  stain  while  its  fellow  fails  to  do  so.  In  such  a  case  faulty 
technique  cannot  be  held  responsible,  nor  can  the  variation  be 
attributed  to  differences  in  environment.  Moreover  in  the 
case  of  the  meningococcus  it  has  been  found  to  persist  after 
animal  passage  (McDonald,  1908).  The  variation  would  appear 
to  date  from  the  cell  division  which  constitutes  the  "birth" 
of  the  organism.  Denny  (1903)  observed  the  same  inequality 
in  staining  properties  in  different  segments  of  a  segmented 
form  of  B.  Xerosis. 

Many  other  examples  of  spontaneous  variation  will  be 
found  in  later  pages. 

2.  Differences  in  characters  are  sometimes  associated  with 
differences  in  geographical  distribution.  Thus  Schultz  (1909) 


14  CONDITIONS  MODIFYING  [OH.  n 

found  that  in  Cleveland  U.S.A.,  during  the  12  months  covered 
by  the  investigation,  "barred"  forms  of  the  diphtheria  bacillus 
had  almost  disappeared ;  during  the  same  period,  in  Boston 
and  Providence,  another  observer  noted  that  "barred"  forms 
were  unusually  common  while  "granular"  forms  were  very 
rarely  met  with. 

3.  Many  organisms  after  prolonged  cultivation  on  artificial 
media  display  variation  in  character. 

In  some  of  these  cases  the  length  of  the  period  of  cultiva- 
tion is  not  the  cause  of  the  modification,  it  merely  extends  the 
survey  over  a  large  number  of  generations  and  so  enables  the 
observer  to  detect  variations  spontaneously  occurring. 

In  other  cases  the  length  of  iime permits" natural  selection" 
to  play  its  part  and  produce  modifications  which,  in  a  shorter 
interval,  would  not  have  advanced  far  enough  to  be  apparent. 

In  other  cases,  again,  the  prolonged  exclusion  from  animal 
tissues  does  lead  directly  to  a  modification  in  character  which 
is  proportionate,  as  regards  its  extent  and  its  permanence,  to 
the  duration  of  such  exile,  but  disappears  when  the  organism 
is  again  "passed"  through  the  body  of  an  animal.  This  is  true 
more  particularly  of  the  property  of  virulence  (q.  v.). 

As  an  example  of  the  influence  exerted  by  prolonged 
cultivation  in  modifying  the  character  of  an  organism  may  be 
cited  the  statement  of  Mohler  and  Washburn  (1906)  that 
a  strain  of  bovine  tubercle  bacilli  after  cultivation  for  11  years 
was  found  to  have  become  modified  in  morphological  and 
cultural  characters  to  the  human  type. 

Lentz  (quoted  by  Bahr,  1912)  found  that  a  "Flexner" 
type  of  B.  dysenteriae  after  9  years'  laboratory  cultivation 
completely  lost  the  power  to  ferment  maltose.  Arkwright 
(1909)  mentions  a  strain  of  the  meningococcus  which  when 
first  isolated  did  not  ferment  glucose  but  after  ten  months' 
artificial  cultivation  developed  power  to  do  so.  Rettger  and 
Sherrick  (1911)  describe  the  gradual  loss  of  power  to  produce 
pigment  on  the  part  of  an  old  stock  culture  of  B.pyocyaneus 
after  5  years'  artificial  growth. 

Prolonged  cultivation  in  a  medium  containing  a  particular 
carbohydrate  may  develop  in  a  strain  of  bacteria  the  ability 


CH.  n]        THE  CHARACTERS  OF  BACTERIA  15 

to  ferment  that  carbohydrate.  B.  typhosus  for  example  after 
two  years'  growth  in  a  medium  containing  lactose  acquires  the 
power  to  ferment  this  sugar  (vide  p.  58). 

4.  In  other  cases  again  the  length  of  the  period  of  cultiva- 
tion is  of  less  importance  than  the  conditions  under  which 
such  cultivation  takes  place. 

(a)  Conditions  which  lower  the  vitality  of  a  strain  may 
modify  its  characters.   Such  conditions  include  starvation  (for 
example,  growth  in  pure  water),  acidity  of  the  medium,  want 
of  oxygen,  the  presence  of  antiseptics,  exposure  to  sunlight, 
high  or  low  temperatures,  symbiosis,  etc. 

One  example  will  suffice.  A  strain  of  B.  ruber  of  Kiel  if 
heated  to  a  temperature  just  below  that  known  to  kill  the 
organism  loses  its  power  to  produce  pigment  (Adami,  1892). 

(b)  The  crowding  together  of  the  organisms  on  the  surface 
of  the  medium  may  lead  to  a  diminution  in  pigment  produc- 
tion in  the  staphylococcus  aureus  (Andrewes  and  Gordon, 
1905-6)  and  an  earlier  appearance  of  granular  staining  forms 
of  the  diphtheria  bacillus  (Denny,  1903). 

(c)  The  temperature  at  which  organisms  grow  is  respon- 
sible  for   certain   variations.     Laurent   (1890)   found   that 
a  selected  strain  of  B.  ruber  which  had  grown  for  12  months 
at  a  temperature  of  25° — 35°  C.  without  exhibiting  a  trace  of 
colouration,  yielded  its  characteristic  pigment  when  the  tem- 
perature was  lowered  to  18°  C.  An  apparent  staphylococcus 
"albus"  growing  at  37°  C.  may  become  a  vivid  "aureus"  at 
22°  C.  (Andrewes  and  Gordon,  1905-6). 

The  virulence  of  B.  anthracis  is  greatly  modified  by  growth 
at  43°  C.  and  that  of  B.  diphtheriae  may  be  destroyed  by 
subjection  to  a  similar  temperature  (Hewlett  and  Knight, 
1897). 

Wilson  (1910)  describes  an  atypical  B.  typhosus  which 
fermented  lactose  at  a  temperature  of  22°  C.  but  failed  to  do 
so  at  37°  C.  Coplans  (1909)  found  that  dulcite  was  more  quickly 
fermented  by  certain  colon  bacilli  at  20°  C.  than  at  37°  C. 

Rodet  (quoted  and  confirmed  by  Adami,  Abbott  and 
Nicholson,  1899)  found  that  at  a  temperature  of  45°  C.  B.  coli 
developed  in  a  few  hours  into  long  filaments.  The  same 


16  CONDITIONS  MODIFYING  [OH.  n 

agency  will  abolish  the  power  of  some  bacteria — such  as 
B.  anthracis — to  form  spores,  and  may  modify  the  character 
of  the  colonies  it  forms  (Bairibridge,  1903). 

Bacteria  of  the  paratyphoid  group  agglutinate  much  less 
readily  after  being  heated  (Sobernheim  and  Seligmann,  1910). 

(d)  Differences  in  atmospheric  pressure  may  modify  the 
activity  of  certain  organisms. 

B.  coli  yields  formic  acid  and  gas  from  glucose  at  ordinary 
atmospheric  pressure.  If  the  pressure  is  raised  the  yield  of 
gas  diminishes  but  the  yield  of  formic  acid  increases  (Harden, 
1901). 

A  great  pressure  of  carbon  dioxide  is  said  to  deprive  B. 
anthracis  of  its  power  to  form  spores  though  it  has  no  effect 
on  the  vitality  of  the  organism  (Muir  and  Ritchie). 

Certain  organisms  which  do  not  readily  lose  their  virulence 
on  artificial  media  do  so  rapidly  if  grown  in  an  atmosphere  of 
compressed  air  (ibid.). 

(e)  The  presence  or  absence  of  oxygen  is  another  factor 
of  importance.   Strains  of  B.  typhoid  and  B.  coli  growing  in 
water  maintain  their  viability  better  if  plentifully  supplied 
with  oxygen  (Whipple  and  Mayer,  1906). 

In  the  absence  of  free  oxygen  B.  pyocyaneus  ceases  to 
produce  pigment  (Adami,  1892)  though  the  spirillum  rubrum 
produces  it  more  plentifully  (Muir  and  Ritchie). 

Torrey  (1905)  observed  that  by  alternate  aerobic  and 
anaerobic  culture  a  certain  type  of  dysentery  bacillus  had  its 
power  to  ferment  maltose  greatly  augmented. 

Andrewes  and  Horder  (1906)  found  that  a  certain  strepto- 
coccus which  refused  to  ferment  lactose,  under  ordinary  con- 
ditions of  cultivation,  did  so  readily  when  deprived  of  oxygen. 

Kruse  (quoted  by  Glenn,  1911)  found  that  a  staphylococcus 
which,  similarly,  refused  to  liquefy  gelatin  did  so  at  once 
under  the  same  altered  conditions. 

Anthrax  bacilli  in  the  absence  of  oxygen  may  develop 
torula  zoogleic  forms  (Wood,  1889).  Noguchi  (1910)  discovered 
that  B.  biftdus  communis  only  exhibited  the  bifurcating 
phase  under  anaerobic  conditions  and  in  the  absence  of 
oxygen  became  less  pathogenic. 


CH.  ii]        THE  CHARACTERS  OF  BACTERIA  17 

It  is  well  known  that  B.  diphtheriae  produces  toxins  more 
plentifully  in  a  free  supply  of  air  (Clark,  1910). 

The  bacillus  of  malignant  oedema  is  said  to  lose  virulence 
when  grown  aerobically  (Harass,  1906)  and  that  of  cholera  to 
gain  virulence  when  grown  anaerobically  (Hueppe,  quoted  by 
Adami,  1892). 

Foa  (1890)  describes  how  a  strain  of  the  pneumococcus 
could  by  anaerobic  growth  be  deprived  of  its  property  of 
causing  a  characteristic  inflammatory  oedema  of  the  skin 
when  injected  into  an  animal. 

Wood  (1889)  attributed  the  diminished  infectivity  of 
virulent  organisms  discharged  from  the  bowel  in  many 
diseases  to  the  fact  that  in  the  bowel  they  are  practically 
deprived  of  oxygen. 

(f)  Bright  sunlight  destroys  the  virulence  of  some  patho- 
genic organisms  (Marshall  Ward  and  Blackmail,  1910)  and 
leads  to  the  loss  of  pigmentation  in  others  (Laurent,  1890). 
The  bacterium  mycoides,  on  the  other  hand,  will  only  produce 
its  red  pigment  in  the  dark  (Scholl,  quoted  by  Wood,  1889). 

5.  Exposure  to  the  ultra  violet  rays  has  recently  been 
shown  by  Madame  Henri  (1914)  to  effect  a  startling  change 
in  both  the  morphology  and  the  pathogenicity  of  B.  anthracis. 
Cocci  and  filamentous  forms  were  produced  which  differed 
from  the  original  bacilli  in  their  power  of  retaining  stains, 
of  forming  spores,  of  liquefying  gelatine  and  coagulating  milk, 
and  which  gave  rise,  on  injection  into  an  animal,  to  symptoms 
quite  unlike  those  produced  by  normal  anthrax  bacilli.  The 
new  forms  did  not  revert  after  daily  subculture  for  over  two 
months. 

1  6.  Electrolysis.  Electrolysis  may  produce  changes  in  the 
morphology  and  staining  properties  of  bacteria.  Russ  has 
observed  the  production  of  elongated  forms  of  B.  coli,  with 
altered  reaction  to  Gram's  stain,  in  urine  (within  the  human 
bladder  and  outside  the  body)  as  a  result  of  the  passage  of 
a  galvanic  current  of  T15th  m.a.  strength  for  one  hour.  The 
modification  persisted  for  many  months. 

7.  The  age  of  the  culture  is  of  importance  in  the  case  of 
many  pleomorphic  organisms.  For  example,  B.  megatherium 

D.  2 


18  CONDITIONS  MODIFYING  [CH.  n 

and  B.  subtilis  pass  in  a  few  hours  from  a  bacillary  motile 
stage  with  cilia,  to  one  of  filamentous  growth  preceded  by  the 
casting  off  of  cilia  (Marshall  Ward  and  Blackman,  1910). 

This  factor  influences  the  characters  not  only  of  bacteria 
known  to  be  "pleomorphic"  but  of  others  also.  Young  tubercle 
bacilli  are  said  not  to  be  "acid-fast"  (Hamer,  1900). 

Young  cultures  of  B.  diphtheriae  more  often  show  branched 
and  clubbed  forms  (Kanthack  and  Andrewes,  1905)  and  solid 
staining  bacilli  (Denny,  1903)  than  do  older  cultures;  at  the 
same  time  they  are  unable  to  ferment  glycerine  and  lactose 
though  older  cultures  usually  ferment  both  (Muir  and  Ritchie). 

A  young  culture  of  B.  coli  does  not  yield  indol  (MacConkey, 
1909).  Wood  (1889)  found  that  an  old  culture  of  cholera  failed 
to  liquefy  gelatin  but  did  so  readily  after  subculturing,  and 
that  a  young  culture  of  the  same  organism  was  much  more 
susceptible  to  the  action  of  antiseptics  than  a  younger  one. 

Old  cultures  of  pigment  forming  bacteria  are  often  colour- 
less (Adami,  1892). 

Arkwright  (1909)  found  bacillary  forms  of  the  meningo- 
coccus  in  old  cultures.  The  tubercle  bacillus  also  in  old 
cultures  displays  elongated  and  even  branched  forms. 

8.  The  character  of  the  culture  medium  employed  may 
influence  bacteria  in  many  ways. 

(a)  The  age  of  the  medium.    S.  pyogenes  normally  does 
not  ferment  saccharose,  raffinose  or  salicin,  but  if  old  media  be 
used  this  organism  will  ferment  all  three  substances  (S.  Martin, 
1908-9).    On  the  other  hand  B.  diphtheriae,  which  in  fresh 
beef  serum  gives  its  characteristic  "sugar"  reactions,  fails  to 
do  so  if  this  medium  is  old  (Fisher,  1909). 

(b)  Changes  in  the  reaction  of  the  medium  employed  is  in 
many  cases  accompanied  by  changes  in  the  morphological 
characters  of  organisms  growing  in  it,  bacilli  giving  place  to 
cocci  and  diplococci,  and  vice  versa,  and  pigment  formation 
being  modified  or  lost  (Adami,  1892).   The  reaction  also  affects 
the  vitality  of  many  bacteria  (Wood,  1889),  and  their  virulence 
(Peckham,  1897). 

(c)  The  nature  of  the  medium  is  important.    Individual 
morphology,  the  appearance  of  colonies,  fermenting  power, 


CH.  IT]        THE  CHARACTERS  OF  BACTERIA  19 

indol  formation,  pigment  production,  and  virulence,  all  vary 
with  the  kind  of  medium  used. 

Gordon  (1900-1)  states  that  the  streptococcus  of  scarlatina 
may  form,  on  serum,  rods  which  closely  resemble  B.  diph- 
theriae  but  in  a  liquid  medium  it  grows  in  a  typical  strepto- 
coccal  form. 

B.  diphtheriae  does  not  form  toxins  readily  if  there  is  much 
carbohydrate  present  in  the  medium  (Fisher,  1909). 

Many  media  contain  substances  derived  from  the  living 
body  such  as  serum,  or  blood,  and  to  this  extent  are  "natural" 
rather  than  "artificial"  media  and  the  alteration  in  the 
character  of  organisms  growing  in  them,  particularly  as  regards 
virulence,  is  possibly  to  be  attributed  to  this  factor. 

Penfold  (1914)  mentions  the  fact  that  vaccination  with  a 
plague  strain  grown  on  agar  will  protect  rats  against  itself 
but  not  against  the  same  strain  grown  on  serum. 

If  the  ordinary  artificial  media  are  replaced  by  the  natural 
secretions  of  the  body  the  modifications  in  character  on  the 
part  of  the  organisms  growing  in  them  may  be  even  more 
marked. 

Rosenow  (1912-13)  found  that  a  streptococcus  which 
presented  certain  morphological  and  cultural  characters  on 
ordinary  media,  underwent  a  profound  modification  in  respect 
to  both  as  a  result  of  growth  in  unheated  milk. 

Horrocks  (1911)  found  that  a  strain  of  B.typhosus,  obtained 
from  the  urine  of  a  "carrier,"  lost  virulence  on  ordinary  media 
(broth,  and  agar)  in  a  few  days  but  maintained  it  for  over  a 
year  in  urine. 

Both  in  milk  and  in  urine  B.  eoli  may  form  a  dense  network 
of  branching  filaments  (Re vis,  1908,  Wilson,  1908)  and  give 
atypical  fermenting  reactions  (ibid.). 

In  the  presence  of  saliva  B.  coli  yields  leptothrix  forms 
(Adami,  Abbott  and  Nicholson,  1899),  while  S.  mastitidis  is 
deprived  of  virulence  (Savage,  1908-9). 

Diplococcic  forms  of  B.  coli  occur  in  bile  (Adami,  Abbott 
and  Nicholson),  while  in  ascitic  fluid  the  same  organism 
undergoes  profound  changes  in  respect  both  to  its  morphology 
and  its  fermenting  power  (ibid.). 

2—2 


20  CONDITIONS  MODIFYING  [CH.  n 

Pathological  exudations  influence  the  characters  of 
bacteria  growing  in  them  to  an  even  greater  degree  than 
the  natural  secretions.  This  is  particularly  true  of  virulence 
(vide  p.  77). 

Harris (1901)  examined  15  strains  of  B.  coli  from  u natural" 
sources — such  as  sewage,  water,  milk,  shellfish — and  also 
11  strains  from  "diseased"  sources,  that  is  to  say  from  in- 
flammatory exudations.  Of  the  former,  only  two  were  virulent ; 
of  the  latter  only  one  was  non-virulent. 

Growth  in  water  also  influences  bacteria.  B.  coli  in  river 
water,  where  they  are  practically  deprived  of  proteid  food, 
appear  to  lose  their  power  of  producing  indol  (Peckham, 
1897).  The  same  organism  isolated  from  drinking  water  was 
found  by  Savage  (1904)  to  form  less  typical  colonies  than  when 
isolated  from  sewage  or  faeces,  while  Jenner  (1898)  describes  its 
morphological  appearances  as  being  different,  bacilli  isolated 
from  water  being  less  thick  and  opaque — a  distinction  which 
disappeared  when  the  strain  was  grown  in  milk. 

(d)  The  addition  of  various  chemical  substances,  such  as 
antiseptics,  to  the  media  used  for  cultivation  profoundly 
modifies  the  development  of  bacteria.  In  the  presence  of 
carbolic  acid  typhoid  bacilli  assume  the  form  of  non-motile 
cocci  and  diplococci  (Adami,  1892),  the  bacillus  anthrax  loses 
virulence  and  also  the  power  to  form  spores  (Roux,  1890), 
many  bacteria  no  longer  liquefy  gelatin  (Wood,  1889),  while 
others  lose  their  power  to  ferment  carbohydrates  (Penfoldr 
191  IB).  Under  the  influence  of  antiseptics  B.  prodigiosus 
forms  spirillae  and  ceases  to  produce  pigment  (Wasserzug, 
1888).  The  bacillus  of  blue  pus — normally  a  small  short 
bacillus — yields,  on  the  addition  of  a  trace  of  boric  acid  to 
the  medium,  S-shaped  forms  and  close  spirals,  and  on  the 
addition  of  potassium  bichromate,  long  undulating  filaments 
(ibid.).  The  presence  of  sodium  benzoate  inhibits  gas  pro- 
duction in  the  case  of  B.  coli  (Herter,  1909).  The  addition  of 
glycerine  prevents  the  liquefaction  of  gelatin  by  bacteria 
(Adami,  1892)  and  also  inhibits  the  formation  of  indol  (Wood, 
1889).  A  virulent  B.  diphtheriae  is  promptly  attenuated  by 
the  addition  of  iodine  trichloride  to  the  medium  (Mohler  and 


CH.  n]        THE  CHARACTERS  OF  BACTERIA  21 

Washburn,  1906)  and  a  non- virulent  bacillus  of  Blackleg 
rendered  virulent  by  the  addition  of  lactic  acid  (ibid.).  In- 
creased resistance  to  antiseptics  may  be  developed  by  pro- 
longed exposure  to  their  action  (Rettger  and  Sherrick,  1911 : 
Penfold,  1911  c). 

Many  other  examples  are  given  in  later  pages. 

9.  Unusual  fermenting  properties  on  the  part  of  bacteria 
are  frequently  acquired  after  prolonged  growth  on  special 
sugar  or  peptone  containing  media.    The  length  of  time 
required  varies  in  different  cases,  within  wide  limits.     For 
example,  B.  typhosm  can  be  "trained"  to  ferment  dulcite  in 
less  than  two  weeks  (Penfold,  1910  A)  but  cannot  be  trained 
to  ferment  lactose  in  less  than  two  years  (ibid.).   The  method 
is  not  invariably  successful,  but  this  may  be  due  to  the  fact 
that  the  trial  in  many  cases  is  not  sufficiently  prolonged. 

Unusual  proteolytic  powers  may  be  developed  in  a  strain 
of  organisms  by  an  analogous  process.  Miss  Peckham  (1897) 
induced  the  power  to  form  indol  on  the  part  of  many  strains 
of  B.  typhosm  by  cultivating  them  in  a  medium  rich  in  peptone. 

10.  By  artificial  selection,  through  a  number  of  genera- 
tions, it  is  often  possible  to  develop  variation  in  a  particular 
direction. 

Goodman  (1908)  by  this  method  obtained  from  a  strain  of 
B.  diphtheriae  with  moderate  power  of  producing  acid  in 
dextrose  broth,  two  strains  possessing  respectively  a  greatly 
augmented  and  a  greatly  diminished  power  of  acid  production. 

Rettger  and  Sherrick  (1911)  in  the  same  manner  obtained 
from  a  slightly  pigmented  strain  of  B.  prodigiosus  two  strains 
showing  in  one  case  brilliant  colouration  and  in  the  other 
complete  absence  of  colour.  They  also  obtained  by  selection 
a  strain  of  staphylococcus  aureus  unusually  resistant  to  the 
action  of  corrosive  sublimate. 

Conn  (quoted  Glenn,  191 X)  obtained  by  the  same  method 
strains  of  a  micrococcus  with  high  and  low  powers  of  liquefy- 
ing gelatin. 

This  method,  however,  may  prove  unsuccessful.  Rettger 
and  Sherrick  failed  to  modify  pigment  production  in  B.  ruber 
balticus  and  they  quote  Buchanan  and  Traux  as  having  been 


22  CONDITIONS  MODIFYING  [CH.  n 

unable  to  establish  high  and  low  acid  producing  races  of 
streptococcus  lacticus.  Glenn  (1911)  failed  to  produce  high 
and  low  acid  producing  strains  of  B.  proteus.  Other  observers 
have  failed  in  attempts  by  selection  to  develop  a  particular 
morphological  type  of  the  diphtheria  bacillus  (Clark,  1910) 
and  to  modify  the  agglutination  reactions  of  B.  typhosus 
(Moon,  1911). 

11.  Symbiosis  is  known  to  influence  the  behaviour  of 
bacteria  and  has  in  many  cases  a  marked  effect  on  the  character 
of  one  or  other  of  the  organisms  growing  together.  The 
phenomenon  of  symbiosis  is  a  familiar  one  in  vegetable  life. 
The  individual  struggle  for  existence  is  observed  to  give  place 
occasionally  to  a  permanent  partnership  between  two  organisms 
for  their  mutual  benefit.  An  example  of  such  cooperation  is 
furnished  by  the  Lichens  each  of  which  is  a  dual  organism 
composed  of  a  fungus  and  an  Alga.  Vegetable  life  as  a  whole 
is  dependent  upon  the  activity  of  nitrifying  organisms  in  the 
soil  and  in  some  cases  a  definite  alliance  is  formed  between 
the  two  parties,  as  in  the  case  of  the  Leguminosae  and  the 
nitrifying  bacteria  which  take  up  their  residence  in  the  root 
nodules  of  these  plants. 

All  forms  of  mutual  parasitism  are  in  reality  examples  of 
symbiosis.  One  species  of  bacteria  may,  however,  be  dependent 
for  its  growth  upon  another  species  without  necessarily  being 
parasitic.  Thus  Pasteur  advanced  the  theory  that  aerobic 
bacteria  by  exhausting  the  supply  of  oxygen  gave  anaerobic 
bacteria  a  chance  of  growing. 

Allen  (1910)  found  that  a  strain  of  B.  inflmnzae,  which 
could  not  be  grown  on  ordinary  media,  grew  luxuriantly 
on  sterilised  media  in  which  the  staphylococcus  albus  had 
previously  grown.  Neisser  was  able  to  cultivate  the  same 
bacillus  on  plain  agar  for  several  generations  by  growing  the 
Xerosis  bacillus  with  it,  though  a  dead  culture  of  the  latter 
had  not  the  same  favouring  effect  (Muir  and  Ritchie). 

In  other  cases  the  growth  of  one  species  is  inimical  to  the 
growth  of  another.  B.  typhosus  will  not  grow  in  a  filtered 
broth  culture  of  staphylococcus  albus,  nor  in  that  of  many 
other  organisms  (Freudenriech,  1888).  The  meningococcus  is 


CH.  IT]        THE  CHARACTERS  OF  BACTERIA  23 

inimical  to  the  growth  of  the  Klebs-Loeffler  bacillus  (Smirnow, 
1908). 

Prescott  and  Baker  (1904)  describe  a  similar  antagonism 
between  streptococci  and  B.  coli  and  they  attribute  the 
extinction  of  the  latter  when  the  two  are  grown  together  to 
the  greater  sensitiveness  of  B.  coli  to  the  lactic  acid  produced 
by  both  combatants. 

Klein  (1903-4)  found  that  a  strain  of  B.  typhosm  was 
killed  by  B.  coli  in  the  peritoneal  cavity  and  Horrocks  (1911) 
found  that  the  same  thing  happened  in  water  which  contained 
both  organisms.  Jordan,  Russell  and  Zeit  (1904)  observed 
that  B.  typhosm  quickly  died  out  in  polluted  water. 

Symbiosis  is  observed  to  influence  not  only  the  viability  of 
bacteria  but  their  virulence,  their  morphology,  their  fermenting 
powers,  and  other  characters.  The  presence  of  the  strepto- 
coccus is  said  to  be  inimical  to  the  growth  of  the  diphtheria 
bacillus  (Smirnow,  1908)  but  it  increases  the  virulence  of  the 
latter  during  "passage"  (Muir  and  Ritchie,  1910)  while  on 
artificial  media  it  induces  changes  in  the  morphology  of  the 
bacillus,  "granular"  forms  appearing  earlier  than  in  a  pure 
culture  (Denny,  1903).  Smirnow  (1908)  observed  that  the 
same  bacillus  grown  on  agar  in  the  presence  of  a  bacillus 
isolated  from  an  acute  rhinitis,  assumed  a  coccic  form — 
retaining,  however,  its  virulence  unimpaired.  The  meningo- 
coccus  produced  a  similar  change. 

Lesieur  (1901,  quoted  Clark,  1910)  claimed  that  the  pseudo- 
diphtheria  bacillus  may  assume  the  morphological  characters 
of  the  Klebs-Loeffler  bacillus  as  a  result  of  symbiosis  with 
aurococcus  aureus. 

Horrocks  (1911)  found  that  a  typical  strain  of  B.  typhosus 
lost  its  power  to  ferment  "sugars"  when  grown  in  the  presence 
of  a  strain  of  B.  coli  derived  from  a  typhoid  carrier  (vide 
p.  119). 

In  some  cases  bacteria  growing  together  are  able  to  produce 
results  which  neither  can  do  alone.  For  example,  neither 
B.  coli  nor  B.  dentriftcans  alone  can  reduce  nitrates,  but  if 
allowed  to  act  on  sodium  nitrate  together  they  bring  about  the 
escape  of  free  nitrogen  (Marshall  Ward  and  Blackman,  1910). 


24  CONDITIONS  MODIFYING  [CH.  n 

In  other  cases  the  prolonged  growth  of  two  species  together 
appears  to  produce  no  change  whatever  in  either  of  them. 
Williams  (1902)  grew  a  virulent  streptococcus  and  a  virulent 
diphtheria  bacillus  together,  transplanting  every  three  or 
four  days  for  90  such  "generations"  without  influencing  the 
characters  of  either  organism.  Horrocks  (1911)  grew  B.  typho- 
sus  and  B.  fluorescens  non-liquefaciens  together  for  a  period 
of  four  months.  Examinations  made  at  intervals  of  one  week 
throughout  this  period  revealed  no  alteration  in  the  character 
or  agglutination  properties  of  the  B.  typhosus. 

The  methods  of  studying  the  effect  of  symbiosis  are  various. 
Simultaneous  growth  can  be  studied  by  "sowing"  different 
species  of  bacteria  together  indiscriminately  on  the  surface  of 
the  medium ;  or  distinct  colonies  may  be  grown  on  a  plate  so 
that  at  first  a  considerable  space  intervenes  between  colonies 
of  the  different  species,  the  interval  gradually  lessening  as  the 
colonies  extend  until  it  is  finally  obliterated ;  a  third  method 
is  that  of  "criss-cross "  planting,  the  effects  of  symbiosis  being 
seen  at  the  intersection  of  the  lines  of  growth ;  or  fourthly, 
one  species  may  be  grown  on  the  surface  of  the  medium  and 
another  deep  to  it  in  the  form  of  buried  colonies ;  or  fifthly, 
a  double  celluloid  sac  may  be  utilised  in  which  the  products 
of  bacterial  growth  can  diffuse  from  one  compartment  to  the 
other;  finally,  successive  growth  offers  a  further  means  of 
investigation,  the  medium  being  sterilised  after  the  growth 
of  one  species  and  then  planted  with  the  other. 

12.  The  methods  of  modifying  bacteria  which  remain  to 
be  described  all  involve  the  agency  of  the  living  tissues  and 
may  be  regarded  as  forms  of  parasitism. 

(a)  Transmission  through  the  alimentary  canal  is  said  in 
some  cases  to  bring  about  modifications  in  character.  It  has 
been  suggested  that  the  bacillus  of  Aertryck  may  assume 
the  characters  and  agglutinative  properties  of  B.  enteritidis 
Gaertner  after  transmission  through  the  intestine  of  the 
mouse. 

Bahr  (1912)  found  that  the  fermenting  powers  of  certain 
strains  of  dysentery  bacilli  were  modified  after  they  had  been 
passed  through  the  intestine  of  the  fly,  although  for  nine 


CH.  n]        THE  CHARACTERS  OF  BACTERIA  25 

months  previously  the  strains  had  repeatedly  given  normal 
sugar  reactions. 

(b)  "Passage  "  through  an  animal,  or  series  of  animals — by 
successive  injections  into  the  blood,  or  into  the  peritoneal 
cavity,  and  subsequent  re-cultivation  from  the  heart's  blood 
or  peritoneal  fluid — is  known  to  modify  the  characters  of 
bacteria  in  many  cases. 

Fermenting  power,  by  such  means,  may  be  greatly  modified 
(vide  p.  57).  Virulence,  again,  may  in  this  way  be  markedly 
increased  towards  some  animals  and  diminished  towards  others 
(vide  p.  81). 

It  is  claimed  that  the  pseudo-diphtheria  bacillus  can  be 
converted  into  the  Klebs-Loeifier  bacillus  by  passage  through 
rabbits  (Lesieur,  1901),  or  through  guineapigs  (Ohlmacher, 
1902). 

Adami,  Abbott  and  Nicholson  (1899)  injected  typical  B.  coli 
into  the  circulation  of  a  rabbit  and  obtained  diplococcic  forms 
of  the  organism  from  the  liver  after  death.  They  isolated  from 
ascitic  fluid  in  another  case  similar  diplococci  which  stained 
irregularly  and  were  non-motile,  did  not  ferment  sugars  or 
produce  indol,  and  formed  colonies  on  agar  closely  resembling 
those  of  S.pyogenes.  By  intraperitoneal  passages  through  three 
guineapigs  these  organisms  were  converted  into  typical  B.  coli. 

Schmitt  (1911)  claims  to  have  so  modified  a  strain  of 
B.  paratyphosus  (Fliigge)  by  passage  through  a  calf  that  it 
afterwards  gave  the  agglutinative  reactions  of  B.  enteritidis 
Gaertner. 

Calves,  monkeys,  rabbits,  guineapigs,  rats,  mice  and  birds 
may  all  be  used  for  this  purpose. 

(c)  A  modification  of  the  last-named  method  consists  in 
growing  organisms  in  a  ceUoidin  sac  within  the  body  cavity 
of  an  animal .  Martin  ( 1 898)  increased  the  virulence  of  a  strain 
of  B.  diphtheriae  by  growing  it  in  a  celloidin  sac  in  the  peri- 
toneal cavity  of  a  rabbit. 

(d)  Growth  in  the  living  tissues  of  an  animal  host  is 
another  method  of  inducing  variation.  It  is  only  in  the  animal 
body  that  the  actinomyces  produces  its  characteristic  rays  or 
clubs  (Bowlby  and  Andre wes,  1913). 


26  CONDITIONS  MODIFYING  [CH.  n 

Ohlmacher  (1902)  claimed  to  have  changed  typical  B. 
diphtheriae  into  Hoffmann's  bacillus  by  48  hours  subcutaneous 
growth  in  a  rat  previously  immunised. 

The  "solid-staining"  type  of  B.  diphtheriae  has  been 
inoculated  into  a  guineapig  and  the  "granular"  type  has 
been  recovered  subsequently  from  the  site  of  the  inoculation 
(Denny,  1903). 

Such  a  method  is  not  invariably  successful.  For  example, 
Baldwin  (1910)  grew  the  human  type  of  tubercle  bacillus  in 
the  living  tissues  of  the  cow  for  nineteen  months,  in  the  hope 
of  modifying  its  characters  to  those  of  the  bovine  type,  but 
without  success. 

(e)  During  the  course  of  a  disease  the  organism  responsible 
is  not  infrequently  observed  to  undergo  modification  with 
respect  to  one  or  another  character.  Thus  in  diphtheria,  as 
convalescence  is  reached,  the  "granular"  forms  of  the  bacillus 
give  place  to  "solid-staining"  types  (Gorham,  1901). 

In  the  chronic  stages  of  cerebrospinal  fever  the  meningo- 
coccus  isolated  from  the  spinal  fluid  is  found  to  have  lost  in 
some  cases  its  power  to  ferment  dextrose  (Connal,  1910). 
Ark wright  (1909)  found  bacillary  forms  of  the  meningococcus 
in  the  spinal  fluid  in  several  cases  of  cerebrospinal  meningitis. 

Adami,  Abbott  and  Nicholson  (1899)  isolated  from  the 
ascitic  fluid  in  cases  of  cirrhosis,  strains  of  B.  coli  (already 
described,  vide  p.  25)  possessing  unusual  morphological, 
cultural  and  fermenting  characters.  Similar  variants  of  B.  coli 
were  obtained  from  an  inflamed  gall-bladder. 

Foa  (1890)  injected  the  pneumococcus  into  a  rabbit  and 
after  its  death  isolated  strains  from  the  lung  and  from  the 
spinal  fluid  which  produced  lesions  of  two  distinct  types  when 
injected  into  other  rabbits.  He  proved  by  experiment  that 
the  difference  between  the  two  strains  was  due  to  differences 
in  the  amount  of  oxygen  available  for  them  in  the  lung  and  in 
the  spinal  canal. 

Rosenow  (1912-13)  describes  a  certain  streptococcus, 
isolated  from  cases  of  epidemic  sore  throat  which  exhibited 
unusual  and  distinctive  morphological  and  cultural  characters. 
"The  strains  isolated  from  the  peritoneal  exudate  and  blood 


CH.  n]        THE  CHARACTERS  OF  BACTERIA  27 

showed  them  to  a  greater  degree  than  those  isolated  earlier  in 
the  attack  or  from  the  tonsils  at  the  same  time.  After  cultiva- 
tion on  blood-agar  it  was  noticed  that  the  strains  from  the 
tonsils  soon  lost  any  distinctive  peculiarities,  whilst  those  from 
the  exudate  retained  them  longer."  He  concludes  that  the 
unusual  character  of  the  organism  was  directly  due  to  residence 
in  the  body  fluids  and  was  accentuated  as  the  disease  advanced. 

Leutscher  (1911)  found  that  pneumococci  isolated  from 
the  lung  in  acute  pneumonia  after  the  crisis  were  more  virulent 
than  those  isolated  earlier  in  the  illness. 

(/)  In  some  cases — the  so-called  "carriers"— after  all 
symptoms  of  a  disease  have  subsided,  the  particular  organism 
concerned  resists  all  attempts  to  eradicate  it  and  continues 
for  an  indefinite  period  to  grow  and  multiply  at  the  site  of 
the  original  lesion.  In  such  cases  the  organism  may  become 
modified  in  the  course  of  time.  This  is  true  more  especially 
of  its  virulence,  but  other  characters  may  be  involved.  Wilson 
(1910)  mentions  a  strain  of  B.  typhosm,  isolated  from  the 
urine  of  a  typhoid  carrier,  which  had  acquired  the  power  to 
ferment  lactose. 


CHAPTER  III 

A  CONSIDERATION  OF  THE  EVIDENCE 

BEFORE  discussing  in  detail  instances  of  variation  and  of 
"  transmutation,"  it  is  necessary  to  consider  the  value  of  the 
evidence  offered  in  support  of  them  and  the  possible  sources 
of  error,  in  the  way  of  both  observation  and  deduction. 

1.  First  and  foremost  must  be  considered  the  possibilities 
of  contamination.    A  single  colony,   even  after  repeated 
subculture  and  replating,  may  not  represent  an  absolutely 
pure  culture  and  cannot  be  proved  not  to  contain  a  single 
bacterium  of  another  species,  the  appearance  of  which  in 
greater  numbers  at  a  later  stage  of  the  experiment  might 
suggest  variation. 

The  importance  of  contamination  as  a  source  of  error  is 
so  obvious  that  efficient  precautions  are  taken  in  almost  all 
cases  to  eliminate  it. 

Barber  (1908)  has  described  a  method  by  which  a  strain 
can  be  grown  from  a  single  organism  thus  ensuring  the 
purity  of  the  culture.  By  means  of  a  glass  pipette  possessing 
an  extremely  fine  aperture — no  larger  than  the  diameter  of 
a  yeast  cell — a  single  organism  is  removed  under  the  micro- 
scope from  a  culture  which  has  been  repeatedly  diluted. 

2.  The  original  infection,  however,  with  which  the  in- 
vestigator is  dealing  may  itself  be  a  "  mixed "  one  and  this 
fact  may  be  overlooked  in  two  ways  : 

The  conditions  of  cultivation  may  favour  the  growth  of 
one  organism  and  inhibit  that  of  another,  so  that  the  first 
may  be  present  in  such  overwhelming  preponderance  that 
the  second  is  for  the  time  being  completely  submerged,  as 
it  were,  and  undetected.  If  the  conditions  change,  as  a  result 
either  of  the  activity  of  the  organisms  themselves  or  the 
intervention  of  the  investigator,  the  balance  may  be  restored 
and  may  even  swing  in  the  opposite  direction,  so  that  the 


CH.  in]    A  CONSIDERATION  OF  THE  EVIDENCE     29 

second  organism  now  predominates  in  numbers  to  such  an 
extent  that  the  first  one  is  lost  sight  of.  Such  a  train  of 
events  might  be  misinterpreted  and  the  assumption  made 
that  variation  or  even  transmutation  had  occurred.  Horrocks 
(1911)  investigated  the  urine  of  a  typhoid  carrier  which  "in 
certain  dilutions  always  gave  practically  pure  cultures  "  of 
B.  typhosus,  although  B.  coli  was  present  in  small  numbers. 
When  the  urine  was  diluted,  however,  an  enormous  increase 
in  the  B.  coli  occurred  and  the  B.  typhosus  rapidly  disappeared. 
Klein  (1903-4)  observed  the  same  sequence  of  events  in  the 
peritoneal  cavity  on  injecting  a  strain  of  B.  coli  which 
contained  typhoid  bacilli. 

Smirnow  (1908)  quotes  experiments  in  support  of  the 
opinion  that  streptococci  inhibit  the  growth  of  the  Klebs- 
Loeffler  bacillus,  but  only  for  a  time  and  he  explains  in  this 
way  the  appearance  in  some  cases  of  the  bacillus  in  what,  a 
few  hours  previously,  had  appeared  to  be  an  almost  pure 
culture  of  streptococci. 

Two  other  instances  may  be  given.  The  sputum  of  a 
patient  suffering  from  pneumonia  and  a  swab  from  the  throat 
in  a  case  of  diphtheria  may  contain,  in  addition  to  the  virulent 
organisms  which  cause  these  diseases,  avirulent  organisms 
closely  resembling  them — the  saprophytic  pneumococcus  and 
the  pseudodiphtheria  bacillus  respectively.  The  saprophytic 
bacteria  in  both  cases  will  grow  at  a  temperature  of  20° — 22°  C. 
though  the  virulent  types  are  both  unable  to  do  so.  It  is 
evident  that,  other  things  being  equal,  the  temperature  of 
the  incubator  will  decide  which  of  the  two  types,  the  virulent 
or  the  avirulent,  will  predominate  in  the  culture.  The  other 
one,  although  actually  present,  may  then  easily  be  overlooked 
unless  an  alteration  in  the  temperature  gives  it,  in  turn,  the 
ascendancy.  In  the  latter  event  the  change  in  virulence  and 
in  other  characters  would  have  the  appearance  of  a  "  variation  " 
brought  about  by  change  of  temperature.  It  would  actually 
be  due  to  a  "contamination"  which  had  been  previously 
overlooked. 

A  second  organism  may,  again,  escape  detection  because 
its  recognition  is  made  dependent  upon  some  one  character 


30     A  CONSIDERATION  OF  THE  EVIDENCE    [CH.  in 

alone — such  as  its  morphology  where  naked  eye  and  micro- 
scopic appearances  are  relied  upon,  or  its  virulence  in  the 
case  of  animal  inoculation,  or  its  fermenting  power  when  the 
culture  is  tested  by  being  "put  through  the  sugars."  The 
organism  may  be  atypical  in  respect  to  the  particular  character 
the  observer  depends  upon  for  its  detection,  and  its  subsequent 
discovery  will  then  lead  to  erroneous  conclusions.  A  knowledge 
of  the  extent  to  which  organisms  may  be  atypical  in  one  or 
other  character  is  the  best  safeguard  against  such  an  over- 
sight. 

The  most  thorough  identification  is  demanded  at  the  con- 
clusion of  an  experiment  no  less  than  at  its  commencement,  and 
the  strictest  rules  must  be  observed  before  the  continuity  of 
two  forms  differing  from  each  other  is  regarded  as  established. 
Such  continuity  may  be  impossible  to  prove  even  when  we 
are  dealing  with  a  "  pure  culture."  A  certain  number  of 
organisms  in  a  pure  culture  may  undergo  variation  while  the 
rest  of  the  strain  remain  true  to  type.  From  time  to  time, 
as  the  conditions  of  growth  change,  now  the  variants  may 
predominate  almost  to  the  exclusion  of  the  original  stock, 
and  now  the  original  stock  may  predominate  almost  to  the 
exclusion  of  the  variants,  so  that,  following  the  variation, 
reversion  may  appear  to  take  place,  and  yet  there  may 
actually  be  no  continuity  in  the  latter  case  between  the 
variants  which  are  dying  out  and  the  original  stock  which  is 
again  asserting  itself. 

3.  In  the  living  tissues  the  possibility  of  secondary  in- 
vasion must  be  borne  in  mind.  For  example,  the  leptothrix 
forms  which  McDonald  (1908)  describes  in  the  spinal  fluid 
in  cerebrospinal  fever,  as  this  writer  himself  recognises, 
cannot  be  regarded  as  morphological  variants  of  the  meningo- 
coccus  without  definite  proof  of  identity. 

Again,  the  pathogenic  effects  in  a  given  case  must  not  be 
attributed  to  an  organism  isolated  from  the  tissues  unless 
adequate  proof  is  forthcoming  of  its  being  in  fact  the  cause 
and  not  a  secondary  invader. 

Forbes  in  1903  drew  attention  to  the  frequency  with  which 
diphtheria  bacilli  were  to  be  found  in  the  ear  discharges  of 


CH.  in]    A  CONSIDERATION  OF  THE  EVIDENCE     31 

patients  suffering  from  scarlet  fever.  The  bacilli  were  present 
in  32  out  of  a  series  of  40  cases  examined  and  sometimes 
greatly  outnumbered  the  other  organisms  present. 

Lustgarten's  bacillus  (1884)  in  syphilis  and  Sanarelli's 
Bac.  icteroides  (1897)  in  the  case  of  yellow  fever  may  be 
quoted  as  examples  of  secondary  invaders  to  the  presence  of 
which  diseases  were  wrongly  attributed,  and  many  other 
instances  might  be  given. 

Bacteria  may  be  present  in  healthy  organs.  Ford  (1900) 
examined  the  liver  and  kidneys  of  healthy  animals  after  death 
with  the  most  stringent  precautions  against  contamination 
and  found  that  at  least  80  per  cent,  contained  bacteria  of 
various  kinds. 

Dudgeon  (1908)  states  that  staphylococcus  albus  can  be 
cultivated  from  the  great  omentum  in  many  healthy  animals 
and  quotes  many  examples  to  show  that  pathogenic  organisms 
can  exist  in  the  body  for  long  periods  without  giving  rise  to 
any  symptoms.  Savage  (1907-8)  has  recorded  the  presence  of 
B.  Gaertner  in  the  intestines  of  healthy  young  calves.  Zwich 
and  Weichel  (1910)  found  that  out  of  177  healthy  mice,  28 
contained  B.  Aertryck  in  their  faeces. 

Post  mortem  invasion  must  be  guarded  against,  for  after 
the  death  of  an  animal  secondary  infection  is  extremely  likely 
to  occur.  Dudgeon  and  Sargent  (1907)  record  a  case  of 
pneumococcal  peritonitis  in  man,  in  which  the  peritoneal 
exudate  one  hour  after  death  gave  a  pure  culture  of  pneu- 
mococci,  whereas  26  hours  later  B.  coli  alone  could  be 
recognised  in  the  same  exudate. 

4.  The  repetition  of  an  experiment  with  an  identical 
result  as  regards  the  variation  produced  is  valuable  con- 
firmatory evidence,  particularly  in  the  hands  of  different 
investigators.    Inability  to  repeat  the  phenomenon,  though 
by  no  means  disproving  its  original  occurrence,  does  to  some 
extent  discredit  it. 

5.  The  constancy  of  the  new  feature,  particularly  on 
subculture,  is  of  importance  both  as  enabling  one  to  exclude 
various  errors  of  observation  and  as  indicating  the  funda- 
mental character  of  the  change.  On  the  other  hand  a  tendency 


32     A  CONSIDERATION  OF  THE  EVIDENCE    [CH.  in 

to  revert  more  or  less  quickly  to  the  original  type  on  the 
removal  of  the  modifying  influence  indicates  racial  stability 
in  character  and  minimises  the  significance  of  the  modification. 
This  aspect  of  the  problem  will  be  referred  to  later  (vide  p.  144). 
It  is  necessary,  however,  to  emphasise  at  this  point  the  danger 
of  assuming  a  change  in  character  to  be  permanent  because 
reversion  has  not  occurred  within  a  certain  period,  even  a 
lengthy  one.  A  strain  of  B.  ruber,  for  example,  may  show  no 
trace  of  colour  for  12  months  together  under  certain  conditions 
and  yet  retain  undiminished  its  power  to  produce  pigment 
in  more  favourable  circumstances  (Laurent,  1890).  In  other 
cases  reversion  occurs  without  any  modification  in  the  con- 
ditions of  growth  but  apparently  spontaneously  and  this  after 
long  periods  of  time  have  elapsed. 

6.  The  necessity  for  perseverance  in  following  a  particular 
line  of  investigation  needs  no  less  emphasis.    Twort  (1907) 
took  two  years  to  train  a  particular  strain  of  B.  typhosus  to 
ferment  lactose — a  result  which  Penfold  (1910  A)  failed  to 
achieve  in  the  case  of  over  a  dozen  strains  after  a  15  months' 
trial.   Coplans  (1909)  grew  a  strain  of  B.  tetani  on  a  gelatin 
medium  for  90  days  before  liquefaction  occurred.   Eyre  and 
Washbourn  (1899)  found  that  to  raise  a  particular  strain  of 
avirulent  saprophytic  pneumococci  to  full  virulence  by  animal 
"passage"   no  less   than   53    successive   inoculations  were 
required.     Goodman  (1908)  in  his  attempts  to  modify  by 
artificial  selection  the  acid  production  in  a  strain  of  diphtheria 
bacilli,  made  18  transfers  before  any  result  was  perceptible. 

In  all  these  cases,  if  the  experiments  had  concluded  earlier, 
negative  results  might  have  been  obtained  and  a  claim  based 
on  this  evidence  for  stability  in  character  which  a  more 
prolonged  investigation  would  have  shown  not  to  be  justified. 

7.  Faultless  technique  is  essential  to  accuracy  in  results. 
In  carrying  out  agglutination  tests,  for  example,  the  utmost 
care  and  patience  is  demanded  even  from  a  practised  observer 
if  his  conclusions  are  to  be  of  any  real  value,  and  much  of 
the  confusion  which  at  present  exists  on  the  subject  of 
agglutination  is  no  doubt  to  be  attributed  to  bad  workman- 
ship. 


CH.  in]    A  CONSIDERATION  OF  THE  EVIDENCE      33 

A  ccurate  observation  is  of  no  less  importance.  For  example, 
the  particular  constituent  of  the  bacterial  protoplasm  which 
retains  a  stain  may  be  unevenly  distributed  throughout  the 
cell.  A  "  solid-staining  "  type  of  bacillus  may  thus  give  rise 
to  one  exhibiting  "polar  staining"  as  in  the  Klebs-Loeffler 
bacillus  and  also,  under  certain  conditions,  B.  coli  and  B. 
typhosus.  If  the  demarcation  between  the  staining  and  the 
non-staining  material  be  very  definite  a  bacillus  showing 
polar  staining  may  closely  resemble  a  diplococcus  andconfusion 
arise  unless  careful  observation  be  made. 

A  deceptive  appearance  may  in  the  same  way  be  produced 
by  the  uneven  staining  of  a  bacterial  filament.  Wilson  (1906) 
found  that  B.  coli  under  the  influence  of  urea  developed 
filamentous  forms.  The  staining  material  in  these  filaments 
under  certain  conditions  became  segmented,  although  the 
organism  as  a  whole  showed  no  sign  of  segmentation,  with 
the  result  that  the  filament  presented  the  appearance  of  a 
chain  of  cocci.  Ainley  Walker  and  Murray  (1904)  had  previously 
observed  the  same  phenomenon  in  the  filamentous  forms  of 
B.  typhosus  produced  under  the  influence  of  methyl  violet. 

Treatment  with  silver  nitrate  may  render  more  apparent 
the  division  of  a  diplococcus  or  a  filament  into  individual 
cells. 

8.  In  other  cases  where  the  actual  technique  is  perfect 
and  the  recognised  method  is  carried  out  in  every  detail,  the 
method  itself  may  be  at  fault ;  conflicting  results  in  such 
circumstances  would  not  be  due  to  any  variation  in  the 
character  of  the  organism  concerned  but  to  such  factors  as 
the  composition  of  the  medium,  the  age  of  the  culture,  the 
time- allowance  made  for  a  "positive  result"  to  declare  itself, 
and  so  on. 

A  few  examples  will  suffice  to  show  the  importance  of 
such  factors. 

(a)  The  composition  of  the  medium.  Sugar  containing 
media  may  be  unsuitable  on  account  of  impure  commercial 
sugars  being  used  in  their  preparation  or  from  their  being 
sterilised  in  vessels  made  of  certain  kinds  of  glass  (W.  B.  M. 
Martin,  1911);  they  may  undergo  decomposition  during  the 
D.  3 


34      A  CONSIDERATION  OF  THE  EVIDENCE    [CH.  in 

process  of  sterilisation  or  they  may  deteriorate  if  kept  for 
some  time  before  being  used,  and  failure  to  guard  against 
these  sources  of  error  may  lead  to  discordant  results. 

The  streptococcus  pyogenes  normally  fails  to  ferment  both 
saccharose  and  raffinose  in  broth,  but  it  produces  acidity  in 
old  media  containing  either  of  these  sugars  (Martin,  1908-9). 
Fisher  (1909)  on  the  other  hand  found  that  diphtheria  and 
diphtheroid  bacilli  which  gave  fermentation  tests  readily  in 
fresh  beef  serum,  failed  to  do  so  if  the  serum  were  old. 

This  observer  and  Theobald  Smith  (1899)  both  state  that 
even  virulent  diphtheria  bacilli  may  fail  to  yield  toxin  if  the 
medium  in  which  they  are  growing  contains  more  than  a 
trace  of  sugar ;  while  Williams  (1902)  found  many  strains, 
which  were  non-pathogenic  when  inoculated  from  ordinary 
broth,  were  highly  toxic  when  inoculated  from  serum  culture 
or  ascitic  broth. 

The  neutral  red  reaction  in  the  case  of  B.  coll  not 
infrequently  fails  but  Moore  and  Re  vis  (1905)  claim  that  if 
lactose  is  substituted  for  glucose  in  the  broth  a  positive  result 
is  invariably  obtained  with  this  organism. 

Glenn  (1911)  observes  that  the  acidity  produced  in  a 
medium  by  the  fermentation  of  its  carbohydrate  constituents 
inhibits  the  production  of  indol  and  may  account  for  the 
failure  of  the  indol  test. 

Wood  (1889)  has  stated  that  the  presence  of  glycerine  in 
a  medium  will  prevent  the  liquefaction  of  gelatin  by  organisms, 
not  by  interfering  with  their  power  of  fermentation  but  by 
offering  them  a  pabulum  they  prefer. 

Again,  if  the  medium  used  in  the  case  of  fermentation 
tests  is  itself  markedly  alkaline,  the  production  of  acid  in 
small  quantities  may  be  completely  masked,  since  it  merely 
results  in  a  diminution  in  alkalinity  and  this  requires  special 
means  of  detection.  Miss  Peckham  (1897)  quotes  Timpe  to 
the  effect  that  all  albuminous  bodies  give  an  alkaline  reaction 
to  litmus  and  it  is  well  known  that  alkaline  products  are 
formed  by  the  breaking  down  of  peptone,  so  that  the  use  of 
litmus  as  an  indicator  in  peptone  holding  material  makes  the 
alkaline  reaction  prominent  even  when  a  considerable  quantity 


CH.  in]    A  CONSIDERATION  OF  THE  EVIDENCE      35 

of  free  acid  is  really  formed.  Clark  (1910)  states  that  Hofmann's 
bacillus  produces  slight  but  definite  acidity  in  dextrose  broth 
if  phenol-phthalein  be  used  as  an  indicator,  whereas  if  litmus 
is  used  the  reaction  always  appears  alkaline. 

(b)  The  age  of  the  culture  is  also  a  factor  of  importance. 
MacConkey  (1909)  finds  that  the  indol  reaction  in  the  case 
of  B.  coll  is  not  given  by  a  2  or  3  days'  culture  ;  the  latter 
should  be  nearly  a  week  old  in  order  to  give  a  positive  result. 
A  young  culture   of  B.  diphtheriae  is  unable  to  ferment 
glycerine  and  lactose  though  an  older  culture  will  usually  do 
so  (Muir  and  Ritchie).    An  old  culture  of  cholera  will  not 
liquefy  gelatin  (Wood,    1889).     Graham  Smith  (1906)    has 
pointed  out  that  many  strains  of  diphtheria  bacilli  do  not 
grow  well  in  broth  when  first  isolated  from  the  throat  and 
therefore  do  not  produce  acidity  at  once. 

(c)  The  time  alloivance.     In  the  case   of  many  sugar 
fermenters  an  incubation  period  of  48  or  even  72  hours  is 
required  before  acidity  becomes  apparent,  and  in  the  case  of 
other  organisms  a  similar  "  latent  period  "  may  elapse  before 
the  appearance  of  pigment. 

Still  longer  observation  is  sometimes  necessary.  Klotz 
(1906)  describes  a  coliform  organism  which  did  not  produce 
indol  until  the  20th  day.  Petrusky  (1889-90)  showed  that  in 
the  case  of  B.  typhosus  a  certain  slow  fermentation  of  lactose 
does  take  place  in  litmus  whey  although  the  organism  is 
regarded  as  a  non-fermenter  of  lactose.  Penfpld  (1910  B) 
states  that  the  same  organism  ferments  dulcite — a  property 
usually  denied  to  it — if  the  experiment  is  prolonged  for  2  or 
3  weeks.  Bahr  (1912)  describes  a  dysentery  bacillus  of  the 
"Flexuer"  type  which,  after  4  days  incubation,  only  fer- 
mented mannite  but  fermented  maltose  and  saccharose  also, 
after  15  days'  incubation.  Wilson  (1910)  states  that  he  has  fre- 
quently isolated  bacilli  from  the  intestine  which  required  from 
9  to  21  days  to  produce  acidity  in  lactose  litmus  broth  and 
several  more  days  to  produce  gas. 

In  other  cases  the  change  in  the  reaction  is  reversed  after 
an  interval.  Thus,  Bahr  mentions  another  "Flexner"  bacillus 
which  produced  a  feeble  acid  reaction  in  mannite  at  the  end 

3—2 


36     A  CONSIDERATION  OF  THE  EVIDENCE    [CH.  m 

of  24  hours  but  after  ten  days  incubation  gave  a  definitely 
alkaline  reaction. 

It  is  obvious  from  these  facts  that  the  question  of  the  time 
allowance  is  of  great  importance.  This  point  will  be  considered 
further  in  connection  with  variations  in  fermenting  power 
(vide  p.  66). 

9.  Finally,  pathological  research  and  clinical  observation 
must  go  hand  in  hand.  The  former,  if  it  is  divorced  from  the 
latter,  is  beset  with  dangers. 

A  certain  patient's  blood,  in  the  laboratory,  may  give  at 
one  time  a  positive  Widal's  test  and  at  another — some  weeks 
later — fail  to  do  so.  The  knowledge  that  on  the  first  occasion 
the  patient  was  the  subject  of  jaundice  would  suggest  a  simple 
explanation  (Griinbaum,  1896)  for  a  phenomenon  otherwise 
difficult  to  elucidate. 

Similarly  the  knowledge  that  sodium  benzoate  was  being 
administered  to  a  patient  suffering  from  cystitis  would  afford 
an  explanation  of  the  fact  that  the  strain  of  B.  coli  contained 
in  the  urine  of  the  patient  showed  a  greatly  diminished  power 
of  gas  production  in  dextrose  (Penfold,  1911  A). 

Again,  altered  pathogenicity  may  be  falsely  attributed  to 
a  strain  of  organisms  if  clinical  observation  is  neglected. 
A  certain  disease  may  be  latent  in  a  patient — that  is  to  say, 
present  without  giving  rise  to  any  noticeable  symptoms.  The 
constitutional  disturbances  arising  from  infection  by  the 
organism  in  question  may  "light  up  "this  pre-existing  disease 
and  the  symptoms  of  the  latter  then  be  incorrectly  credited 
to  the  invading  organism. 


CHAPTER  IV 

VARIATIONS  IN  MORPHOLOGY 

VARIATIONS  in  morphology  will  be  considered  under  three 
heads:  (A)  zoogleic  forms,  (B)  individual  organisms,  (C) 
colonies. 

A.    ZOOGLEIC  FORMS. 

One  remarkable  feature  of  the  bacteria  or  schizomycetes 
is  the  tendency  they  show  when  multiplying  to  become  massed 
together,  not  indiscriminately  but  in  an  orderly  arrangement, 
to  form  "zoogleae."  These  forms  display  an  extraordinary 
diversity  of  shape  and  structure.  Thus,  a  single  bacillus — as 
a  result  of  alternate  elongation  and  division  in  a  transverse 
plane— may  give  rise  to  a  long  filament  consisting  of  a  row  of 
cylindrical  cells  placed  end  to  end.  In  other  cases  a  number 
of  organisms  may  be  crowded  together  in  a  round  gelatinous 
mass,  their  swollen  cell  walls  fusing  to  form  a  mucilaginous 
matrix  in  which  they  lie  embedded  for  an  indefinite  period. 

The  shape  and  structure  of  these  zoogleae  are  not  fortui- 
tous but  appear  to  be  designed  in  many  instances  to  attain 
some  definite  object  of  advantage  to  the  organism,  and  may 
thus  form  a  stage  in  its  life  history.  This  is  the  case  for 
example  in  the  Bacterium  raditicola,  the  nitrogen-fixing 
organism  found  in  the  nodules  on  the  roots  of  leguminous 
plants.  It  first  enters  the  root  hair  from  the  soil;  it  then 
assumes  a  filamentous  form  and — in  a  manner  comparable  to 
the  downward  growth  of  the  pollen  tube  from  the  stigma  to 
the  ovary — pushes  its  way  along  the  interior  of  the  hair  as 
a  long  slimy  thread  until  it  penetrates  the  tissues  of  the  root 
itself. 

Again  the  Beggiatoa  versatilis,  a  vegetation  often  seen  at 
the  mouth  of  drain  pipes,  may  be  observed  to  send  out  from 
a  whitish  gelatinous  ground  mass,  long  oscillating  filaments 


38  VARIATIONS  IN  MORPHOLOGY         [CH.  iv 

which  emerge  after  sundown  and  the  next  day  split  up  into 
innumerable  little  bacteria  rods  (Kerner  and  Oliver). 

The  zoogleae  in  which  bacteria  are  massed  together  are  to 
be  regarded  as  a  resting  stage  in  their  life  history.  The  swollen 
envelope  or  matrix  in  which  they  are  embedded,  and  which 
in  some  cases  becomes  hard  and  chitinous,  being  protective  in 
character. 

It  is  important  however  to  recognise  the  fact  that  these 
zoogleae  are  merely  conglomerations  of  a  number  of  organisms 
and  are  not,  strictly  speaking,  individuals  themselves.  Too 
great  stress  must  not  be  laid,  therefore,  on  their  formation 
and  the  changes  they  undergo  as  evidence  of  variation  on  the 
part  of  the  individuals  composing  them. 

A  regiment  of  soldiers  during  manoeuvres  is  composed  of 
a  number  of  individuals,  all  of  the  same  kind,  comparable  to 
pathogenic  bacteria.  It  assumes  various  forms  from  time  to 
time — that  of  serried  ranks  when  marching,  a  filamentous  form 
when  advancing  in  single  file,  a  "square"  when  awaiting  a 
cavalry  charge  and  yet  another  appearance  during  its  resting 
stage  when  bivouacked  for  the  night.  Again,  a  mass  meeting 
or  "demonstration"  of  coal  miners,  composed  of  a  different 
type  of  individual — all  again  of  the  same  kind,  comparable 
to  a  harmless  pigment-producing  organism — shows  quite  a 
different  formation,  namely  that  of  an  irregularly  shaped  crowd, 
the  units  of  which  are  arranged  somewhat  concentrically. 
There  is  sometimes  a  tendency  to  the  formation,  at  the  peri- 
phery, of  smaller  collections  or  nodules  showing  a  similar 
concentric  arrangement.  There  is  a  constant  tendency  on  the 
part  of  a  regiment  or  a  miners'  "demonstration,"  wherever  we 
find  them,  to  reproduce  exactly  the  forms  described  as  typical 
of  each.  A  regiment  of  soldiers  or  a  crowd  of  pitmen  may  be 
regarded  as  a  separate  entity  in  one  sense,  but  neither  is  an 
individual  in  the  sense  that  a  tree,  composed  of  vegetable  cells, 
is  one.  There  is  no  interdependence  of  one  part  upon  another 
in  a  body  of  troops  or  a  crowd.  They  are  temporary  and  can 
be  dispersed,  the  individual  units  surviving  though  separated 
from  each  other.  Moreover  the  forms  they  assume  are  not 
invariable.  A  regiment  of  soldiers,  if  a  certain  controlling 


CH.  iv]         VARIATIONS  IN  MORPHOLOGY  39 

influence  is  removed,  or  in  response  to  a  particular  stimulus- 
such  as  the  attraction  of  a  boxing-match — may  assume  the 
form  of  an  irregular  crowd  concentrically  arranged.  A  certain 
controlling  influence  in  the  case  of  the  miners,  or  a  common 
spontaneous  impulse,  may  result  in  their  marching  in  military 
formation.  In  other  words,  a  collection  either  of  the  pathogenic 
organisms  or  of  the  harmless  pigment  producers,  may  assume 
temporarily  a  formation  rightly  regarded  as  characteristic  of 
the  other;  but  we  should  be  mistaken  in  supposing  on  this 
account  that  the  soldiers  were  being  transformed  into  miners, 
or  vice  versa. 

The  development  of  zoogleic  forms  may  occur  spontane- 
ously, or  it  may  be  brought  about  artificially. 

I.    ZOOGLEIC  FORMS  OCCURRING  SPONTANEOUSLY. 

These  may  represent  a  regular  phase  in  the  life  history 
of  the  organism;  on  the  other  hand,  they  may  occur  quite 
irregularly  as  an  occasional  variation — either  in  cultures  on 
artificial  media  or  in  the  living  tissues — in  which  case  one  must 
regard  the  change  as  representing  a  phase  in  the  life  history 
of  the  organism  at  an  earlier  stage  in  its  evolution. 

Perhaps  the  earliest  account  of  zoogleic  forms  occurring 
in  the  life  history  of  a  micro-organism  was  that  given  by 
Ray  Lankester  in  1873,  with  reference  to  the  non-pathogenic 
Bacterium  rubescens.  The  units  of  this  bacterium  were 
observed  to  become  aggregated  into  a  multitude  of  forms, 
protean  in  their  variety — stellar,  globose,  massive,  arborescent, 
eaten ular  (or  chain-like),  reticular,  tessellate  and  so  on.  (Dia- 
grams of  each  of  these  forms  are  appended  to  the  original 
article.) 

The  tubercle  bacillus  indicates  its  relationship  to  the 
streptothrices  by  forming  in  old  cultures  a  branching  filament, 
sometimes  with  "clubbed"  ends,  while  in  the  living  tissues, 
under  certain  conditions,  it  gives  rise  to  a  radiating  structure 
similar  to  that  of  the  actinomyces  (Muir  and  Ritchie). 

The  bacillus  of  glanders,  similarly,  on  artificial  culture  may 
exhibit  short  filamentous  forms,  and  under  certain  conditions 


40  VARIATIONS  IN  MORPHOLOGY         [CH.  iv 

in  the  living  tissues  show  branching  filaments  and  "clubbing" 
(ibid.). 

The  Klebs-Loeffler  bacillus  in  young  cultures,  on  serum  and 
agar-agar,  likewise  shows  clubbed  and  branched  forms. 

The  bacillus  of  anthrax,  both  on  artificial  media  and  in  the 
living  tissues,  forms  leptothrix-like  chains  or  filaments.  These 
may  be  observed  in  a  three  hours'  culture  of  the  bacillus  in 
a  drop  of  aqueous  humour  (Marshall  Ward  and  Blackman, 
1910). 

Adami  (1892)  describes  B.  typhosm  as  forming  long  fila- 
ments when  grown  on  potato.  Many  observers  have  recorded 
the  same  phenomenon  in  cultures  of  B.  coli.  For  example, 
Ohlmacher  (1902),  Revis  (1908)  and  Wilson  (1908)  isolated 
leptothrix  forms  of  B.  coli  from  the  heart's  blood  in  a  case 
of  septicaemia,  from  milk  and  from  urine  respectively,  the 
organism  in  each  case  forming  a  dense  network  of  branching 
filaments. 

Ritchie  (1910)  isolated  leptothrix  forms  of  B.  influenzae 
from  the  cerebrospinal  fluid  in  certain  cases  of  meningitis,  and 
similar  forms  of  the  "pseudo-influenza"  bacillus  from  the  lung 
in  pneumonia.  The  causal  factor  in  two  of  these  instances— 
B.  coli  isolated  from  urine  and  B.  influenzae  from  the  spinal 
fluid  in  meningitis — was  probably  the  presence,  in  both  these 
fluids,  of  urea.  Connal  (1910)  showed  that  in  meningitis  the 
spinal  fluid  may  contain  as  much  as  "5  per  cent,  of  urea,  and 
Wilson  (1906)  showed  that  urea  provokes  the  development  of 
leptothrix  forms  in  many  organisms. 

II.    ZOOGLEIC  FORMS  ARTIFICIALLY  PRODUCED. 

1.  The  addition  of  various  chemical  substances  to  the 
culture  medium  in  which  an  organism  grows,  leads  to  the 
development  in  many  cases  of  leptothrix  forms. 

Peju  and  Rajat  (quoted  by  Wilson,  1910)  observed  that 
salts,  and  Almquist  (ibid.)  noted  that  seivage,  had  this  effect  on 
B.  typhosm.  Walker  and  Murray  (1904)  showed  that  certain 
dyes,  particularly  methyl  violet,  had  the  same  action  on 
B.  typhosm,  B.  coli  and  the  cholera  organism.  Wilson  (1906), 
by  adding  urea  to  the  culture  media,  obtained  leptothrix  forms 


CH.  iv]         VARIATIONS  IN  MORPHOLOGY  41 

of  B.  typhosus,  B.  coli,  B.  pyocyaneus,  B.  enteritidis  Gaertner, 
and  B.  pneumoniae  Friedlander ;  Adami,  Abbott  and  Nichol- 
son, by  the  addition  of  human  saliva,  or  a  trace  of  bile, 
obtained  the  same  result  with  B.  coli.  Growth  in  an  acid 
lactose  containing  medium  also  developed  filamentous  forms 
of  this  organism.  They  quote  Schmidt's  observation  that 
growth  in  caustic  soda  broth  had  the  same  effect. 

Adami  (1892)  observed  that  B.  pyocyanem  took  the  form 
of  a  filament  or,  in  some  cases,  developed  into  close  spirals  and 
S-shaped  forms  under  the  influence  of  /9.  naphthol,  alcohol, 
potassium  bichromate,  boric  acid ;  and  Pakes  (1901)  noted  that 
the  nitrates  of  sodium,  potassium,  ammonium,  and  lithium 
developed  in  the  same  organism  filamentous  forms  which 
showed  spurious  branching  and  resembled  a  cladothrix. 

Wasserzug  (1888)  found  that  B.  prodigiosus  formed  long 
bacilli  and  spirilla  if  grown  in  the  presence  of  antiseptics. 
Tartaric  acid  had  the  same  effect  and  by  prolonged  growth 
in  media  containing  this  acid  and  subjection  to  a  temperature 
of  50°  C.  subsequently  for  a  few  minutes,  a  race  of  long  bacilli 
was  obtained  which  retained  its  new  character  "permanently" 
—that  is  to  say  on  its  return  to  ordinary  media. 

2.  The  formation  of  zoogleae  may  be  provoked  by  alteration 
in  temperature.    Rodet  (quoted  and  confirmed  by  Adami, 
Abbott  and  Nicholson,  1899)  found  that  a  culture  of  B.  coli 
at  a  temperature  of  44-45°  C.  developed  within  a  few  hours 
very  long  filaments. 

3.  The  absence  of  oxygen  may  have  the  same  effect.  Wood 
(1889)  observed  "torula"  forms  of  the  cholera  bacillus  when 
grown  in  bouillon  anaerobically.    Noguchi  (1910)  found  that 
B.  bifidus  communis  only  exhibited  its  bifurcating  phase  in 
anaerobic  culture. 

4.  Exposure  to  the  ultra  violet  rays  leads  to  the  formation 
of  long  filaments  in  B.  anthracis  (Henri,  1914). 

5.  Growth  in  the  animal  body  develops  on  the  part  of  the 
actinomyces  its  characteristic  rays  or  clubs.   These  are  not 
seen  in  artificial  cultures  (Bowlby  and  Andre wes). 


42  VARIATIONS  IN  MORPHOLOGY         [OH.  iv 

B.    MORPHOLOGICAL  VARIATIONS  IN  INDIVIDUAL 
ORGANISMS. 

Morphological  variations  in  individual  bacteria  may  occur 
as  normal  phases  in  its  life  history,  or  they  may  develop  in 
response  to  changes  in  their  environment. 

I.    PLEOMORPHISM  IN  THE  LIFE  HISTORY. 

While  many  bacteria  are  only  known  under  certain  forms 
and  are  regarded  as  a  micrococcus,  a  bacterium,  a  bacillus  or 
a  spirillum,  others  are  known  which  in  the  course  of  their 
development  pass  through  several  such  forms  and  are  called 
"pleomorphic." 

The  non-pathogenic  Bacterium  rubescens  already  men- 
tioned (Ray  Lankester,  1873)  affords  a  good  example  of  the 
various  phases  an  individual  organism  may  pass  through  in  its 
life  history — forms  described  as  spherical,  biscuit-shaped,  rod- 
like,  filamentous  and  acicular  succeeding  each  other  in  turn1. 

Amongst  pathogenic  organisms  that  of  cholera  affords  a 
good  example,  the  characteristic  comma-shaped  vibrio  or 
spirillum  giving  place  to  a  coccus  or  to  a  straight  thread 
(Haffkine,  1895). 

In  old  cultures  of  the  meningococcus  bacillary  forms  make 
their  appearance  (Arkwright,  1909).  Young  cultures  of  B.  coli 
show  not  only  typical  bacilli  but  also  small  oval  rods  and  tiny 
coccus-like  forms  (Gordon,  1897).  In  very  young  cultures  of 
B.  diphtheriae  "solid"  types  largely  predominate,  but  in  a  few 
hours  these  give  place  to  "granular"  types  (Denny,  1903). 

The  variations  in  morphology  displayed  by  B.  diphtheriae 
are  many  of  them  so  characteristic  as  to  be  of  value  in  the 
identification  of  the  organism,  and  with  this  object  have 
been  classified  by  Westbrook,  Wilson  and  McDaniel  (1900). 
Similarly  Gordon  (1900-1)  observed  that  the  streptococcus 
of  scarlatina  was  characterised  by  a  tendency  to  take  the  form 

1  Miss  M.  C.  W.  Young  has  recently  reported  some  observations  of  great 
interest  in  this  connection,  revealing  a  similar  pleomorphism  in  bacteria  which 
extended,  in  her  experiments,  over  a  cycle  of  14  days.  (Brit.  Med.  Journ.  1914, 
11,  p.  710.) 


CH.  iv]         VARIATIONS  IN  MORPHOLOGY  43 

of  a  spindle  or  rod,  in  which  case  it  was  difficult  to  distinguish 
it  from  B.  diphtheriae.  At  the  same  time  its  tendency  to 
assume  a  bacillary  form  afforded  a  valuable  means  of  distin- 
guishing it  from  S.  pyogenes. 

II.     MORPHOLOGICAL  VARIATIONS  DUE  TO  ENVIRONMENT. 

1.  These  maybe  associated  with  differences  in  geographi- 
cal distribution.    For  example,  Schultz  (1909)  found  that  in 
Cleveland,  U.S.A.,  during  the  twelve  months  covered  by  the 
investigation,  "barred"  forms  of  the  diphtheria  bacillus  had 
almost  disappeared;  during  the  same  period  in  Boston  and 
Providence  another  observer  noted  that  "barred"  forms  were 
unusually  common  while  "granular"  forms  were  very  rarely 
met  with. 

2.  Prolonged    cultivation   may   influence    morphology. 
Mohler  and  Washburn  (1906)  found  that  bovine  tubercle 
bacilli  after  11  years'  cultivation  had  become  changed  into  the 
human  type. 

3.  The  crowding  together  of  colonies  on  the  surface  of  the 
medium  also  influences  morphology.    In  cultures  of  B.  diph- 
theriae, under  these  conditions,  the  change  from  the  "solid"  to 
the  "granular"  type  takes  place  much  earlier  than  usual 
(Denny,  1903). 

4.  Changes  in  the  medium  employed  may  lead  to  changes 
in   morphology.     Gordon    (1900-1)    published    photographs 
showing  that  the  streptococcus  associated  by  Klein  and  him- 
self with  scarlet  fever  may  form,  on  serum,  rods  which  closely 
resemble  the  diphtheria  bacillus,  though  in  a  liquid  medium 
it  grows  in  typical  streptococcal  form.  This  fact  may  afford  an 
explanation  of  the  observations,  recorded  by  Duncan  Forbes 
(1903)  and  others,  as  to  the  prevalence  of  B.  diphtheriae  in 
the  ear  discharges  of  patients  suffering  from  scarlet  fever, 
without  giving  rise  to  symptoms  of  diphtheria  and  uninfluenced 
by  antitoxin. 

Ohlmacher  (1902)  and  other  observers  have  called  attention 
to  the  fact  that  streptococci  from  the  throat  in  cases  of 
tonsilitis  may,  on  Loeffler's  serum,  assume  the  form  of  bacilli 
closely  simulating  B.  diphtheriae. 


44  VARIATIONS  IN  MORPHOLOGY         [CH.  iv 

Rosenow  (1912-13)  describes  a  streptococcus  which  de- 
veloped unusual  morphological  and  cultural  characters  as  the 
result  of  growth  in  unheated  milk. 

B.  coli  in  ascitic  fluid  and  in  bile  may  assume  diplococcic 
form  (Adami,  Abbott  and  Nicholson,  1899).  Jenner  (1898) 
found  that  B.  coli  isolated  from  water  was  less  thick  and 
opaque  than  normal  B.  coli,  this  distinction  disappearing, 
however,  after  growth  in  milk. 

Changes  in  the  reaction  of  the  medium  may  bring  about 
changes  in  morphology,  bacilli  giving  place  to  cocci  and  diplo- 
cocci  and  vice  versa  (Adami,  1892). 

5.  The  addition  to  the  medium  of  various  cliemical  sub- 
stances influences  morphology.   The  presence  of  urea  converts 
Micrococcus  prodigiosus  into  a  bacillus  (Wilson,  1906),  and 
Bacillus  Pestis  into  a  coccus  grouped  singly  or  in  pairs  or  in 
short  chains  (ibid.).    B.  enteritidis  Gaertner  on  urine-agar 
develops  into  a  coccus  (ibid.),  while  B.  typhosus  and  B. 
pyocyaneus  grown  in  carbolic  acid  (1  in  600),  and  creosote 
(1  in  1000),  assume  the  forms  of  non-motile  cocci  or  diplococci 
(Adami,  1892). 

Deceptive  appearances  are  sometimes  produced  by  the 
unequal  distribution  of  the  staining  material  in  an  organism 
under  conditions  such  as  those  we  are  discussing.  A  bacillus 
may  under  the  microscope  appear  to  be  a  diplococcus  and 
a  filament  resemble  closely  a  chain  of  cocci. 

Haslam  (1898)  found  that  the  shape  of  B.  coli  communis 
depended  upon  the  composition  of  the  medium  in  which  it  was 
growing.  If  the  composition  of  the  medium  were  changed 
every  24  or  48  hours  the  shape  of  the  organism  changed  with 
it.  He  found  that  the  bacillus  was  longest  (in  proportion  to 
its  breadth)  in  media  rich  in  nitrogenous  substances,  such  as 
proteid  and  ammonium  tartrate,  and  shortest  in  glucose  media 
to  which  little  of  such  nitrogenous  material  had  been  added. 

6.  Exposure  to  ultra  violet  rays  in  the  case  of  B.  anthracis 
has  been  shown  to  change  the  bacilli  to  cocci  and  diplococci 
(Henri,  1914). 

7.  Electrolysis.    Electrolysis  may  produce  changes  in  the 
morphology  and  staining  properties  of  bacteria.     Russ,  for 


CH.  iv]         VARIATIONS  IN  MORPHOLOGY  45 

example,  has  noted  the  production  of  elongated  forms  of  B.  coli 
in  urine,  with  altered  reaction  to  Gram's  stain,  as  a  result  of 
the  passage  of  a  galvanic  current  of  ^th  rn.a.  strength  for 
one  hour.  The  modification  was  produced  in  B.  coli  present 
in  the  human  bladder  in  a  case  of  cystitis  and  also  in  a  speci- 
men of  urine  outside  the  body,  and  it  persisted  for  many 
months. 

8.  Symbiosis  may  affect  morphology.     The  presence  of 
streptococci  in  a  young  culture  of  B.  diphtheriae  hastens  the 
appearance  of  "granular"  types  of  the  latter  (Denny,  1903). 
Smirnow  (1908)  found  that  symbiosis  of  B.  diphtheriae  on 
culture  media  with  (a)  a  streptococcus,  (b)  the  meningococcus, 
and  (c)  an  unidentified  bacillus  derived  from  a  case  of  acute 
rhinitis,  led  in  all  three  cases  to  the  appearance  of  coccoid 
involution  forms  of  the  diphtheria  bacillus.    The  experiment 
in  the  case  of  the  unidentified  bacillus  was  repeated  in  another 
way,  the  two  organisms  being  grown  in  the  two  compartments 
of  a  double  celloidin  sac  which  was  inserted  into  the  peritoneal 
cavity  of  a  rabbit.   In  the  place  of  the  diphtheria  bacillus  he 
found  a  Gram-positive  coccus  which,  however,  on  Loeifler's 
blood  serum  reverted  in  24  hours.   A  repetition  of  the  experi- 
ment gave  exactly  the  same  result. 

Lesieur  (1901,  quoted  by  Clark,  1910)  claimed  that  the 
pseudo-diphtheria  bacillus  may  assume  the  morphological 
characters  of  the  Klebs-Loeffler  bacillus  as  a  result  of 
symbiosis  with  aurococcus  aureus. 

9.  Growth  in  the  living  tissues  will  sometimes  modify  the 
morphology  of  organisms.   Gorham  (1901)  observed  that,  as 
convalescence  from  diphtheria  advances,  "granular"  types  of 
the  bacillus  give  place  to  "solid"  types,  and  he  attributes  the 
change  to  the  action  of  the  body  fluids  of  the  now  immune 
patient. 

Adami,  Abbott  and  Nicholson  (1899)  describe  forms  of 
B.  coli,  isolated  from  the  liver  in  normal  and  diseased  animals 
(cow,  sheep,  rabbit,  guineapig)  and  in  man,  which  resembled 
diplococci  in  many  cases,  and  in  others  short  chains  of  three 
or  four  cocci.  Similar  forms  were  obtained  from  the  bile  and 
from  ascitic  and  peritoneal  fluids,  and  were  produced  on  adding 


46  VARIATIONS  IN  MORPHOLOGY         [CH.  iv 

guineapig  bile  to  culture  media,  and  they  attribute  the 
modification  to  the  action  of  the  body  fluids.  Further,  they 
injected  B.  coli  into  the  circulation  in  rabbits  and  found 
subsequently  enormous  numbers  of  this  diplococcic  form  of 
the  organism  in  the  endothelial  cells  lining  the  hepatic  vessels 
and  also  in  the  cells  of  the  liver,  in  the  bile  and  in  the  kidneys. 
These  were  detected  within  30  to  60  minutes  of  the  injection. 
In  some  instances  the  modification  was  so  marked  that  it  was 
not  possible  by  passage  or  other  means  to  obtain  complete 
reversion  to  type.  In  other  cases  after  passage  through 
guineapigs  reversion  took  place.  The  change  was  associated 
with  irregular  staining,  and  with  loss  of  motility,  of  fermenting 
power  and  of  power  to  produce  indol.  They  found  that 
B.  typhostts  underwent  a  similar  modification  under  the  same 
circumstances. 

Jenner  (1898)  found  that  the  difference  in  morphology 
already  mentioned  in  the  case  of  B.  coli  isolated  from  water 
disappeared  after  passage. 

Ark wright  (1909)  and  other  observers  have  described  a 
micrococcus  closely  resembling  the  meningococcus,  found  in 
almost  pure  culture  in  the  spinal  fluid  in  many  cases  of 
meningitis,  which  is  characterised  by  a  tendency  to  assume  a 
bacillary  form. 

Ohlmacher  (1902)  inoculated  a  guineapig  with  a  long 
"  granular  "  type  of  B.  diphtheriae  but  the  organism  recovered 
later  from  the  site  of  the  inoculation  proved  to  be  of  the 
short  "solid  "  type.  The  experiment  was  twice  repeated  with 
the  same  result.  In  two  other  experiments  with  different 
strains  of  B.  diphtheriae  the  same  observer  found  that  during 
"passage"  through  a  guineapig  the  reverse  change  occurred, 
a  short  "solid"  type  of  organism  being  injected  into  the 
animal  and  a  long  granular  type  recovered  from  the  spleen 
and  liver  after  death. 

Mohler  and  Washburn  (1906)  claim  that,  by  prolonged 
growth  in  the  living  tissues  of  a  suitable  animal  host,  one  type 
of  tubercle  bacillus  can  be  so  modified  with  respect  to  its 
morphological  characters  as  to  become  indistinguishable  from 
another  type.  Baldwin  (1910),  however,  grew  a  strain  of  the 


CH.  iv]         VARIATIONS  IN  MORPHOLOGY  47 

human  tubercle  bacillus  in  the  tissues  of  a  cow  for  19  months 
without  effecting  any  change  in  it. 

Gordon  (1900-1)  found  that  the  tendency  exhibited  by 
S.  scarlatinae  to  assume  bacillary  form  on  certain  media  was 
suppressed  after  passage  through  the  guineapig  but  was 
increased  in  some  cases  after  passage  through  the  mouse. 

C.    VARIATIONS  IN  COLONIES. 

A  given  organism  when  grown  on  the  same  kind  of  medium 
and  under  the  same  conditions  always  tends  to  produce  a 
colony  of  a  particular  size,  form  and  appearance.  Such  a 
colony  is  regarded  as  "  typical "  of  the  organism  in  question 
and  considerable  importance  is  attached  to  its  character  as  a 
means  of  isolating  the  particular  organism  and  identifying  it. 
The  substitution  of  a  macroscopic  for  a  microscopic  appearance 
possesses  such  obvious  advantages  that  the  former  is  frequently 
made  use  of  instead  of  the  latter  and  the  more  constant  its 
features  are  found  to  be,  the  more  reliance  is  placed  upon  it. 
The  question  therefore  arises,  how  far  can  the  appearance  of 
its  colonies  be  trusted  as  a  means  of  identifying  an  organism? 

MacConkey  (1909)  in  speaking  of  colonies  makes  two  state- 
ments. (1)  The  colonies  of  an  organism  may  vary  even  on  the 
same  plate.  (2)  Organisms  of  quite  different  character  may 
produce  colonies  almost  identical.  Both  of  these  statements 
may  be  confirmed  by  reference  to  a  common  organism  such 
as  B.  coli. 

1.  It  is  recognised  that  an  organism  will  yield  different 
types  of  colonies  when  grown  upon  different  kinds  of  media. 
The  character  of  the  colony  depends  upon  the  composition  of 
the  medium.  If,  therefore,  the  material  of  a  culture  plate  or 
tube  should  present  slight  differences  in  composition  in 
different  parts  of  its  surface,  it  is  reasonable  to  expect  slight 
corresponding  differences  in  the  colonies  of  an  organism 
growing  upon  it.  Such  an  explanation  is,  however,  inadequate 
to  explain  the  wide  differences  frequently  observed. 

Savage  (1904)  carried  out  an  elaborate  investigation  in 
order  to  ascertain  to  what  extent  colonies  of  B.  coli  on 
gelatin  conformed  to  the  character  commonly  accepted  as 


48  VARIATIONS  IN  MORPHOLOGY         [CH.  iv 

typical  of  this  organism.  He  examined  72  strains,  derived 
from  half  a  dozen  different  sources.  Out  of  this  number  50 
strains  formed  typical  colonies  but  many  of  them  on  further 
cultivation  gave  rise  to  atypical  colonies,  which  later,  however, 
reverted  to  the  common  type.  The  remaining  22  strains 
(30'5  per  cent.)  all  yielded  atypical  colonies.  Many  of  these 
colonies  bore  no  resemblance  whatever  to  the  common  type 
and  showed  no  tendency  to  revert  to  it ;  moreover,  they 
differed  as  much  from  each  other  as  from  the  typical  colony. 
(Photographs  showing  the  different  appearances  are  to  be 
seen  with  the  original  article.)  One  strain  (No.  160)  replated— 
after  varying  intervals — eight  times  in  the  course  of  several 
months,  gave  rise  to  no  less  than  14  distinct  types  of  colony, 
all  of  them  atypical.  In  other  respects  the  organisms  proved 
to  be  in  every  case'  typical  B.  coli  in  pure  culture.  In  his 
opinion  the  material  from  which  the  organism  was  isolated 
considerably  influenced  the  type  of  colony  formed. 

2.  The  second  statement  is  confirmed  by  the  same  in- 
vestigator who  observed  that  colonies  whose  appearance  was 
absolutely  typical  of  B.  coli,  might  be  composed  of  different 
organisms  altogether.   Many  years  previously  Klein  (1899-00) 
pointed  out  that  it  was  not  safe,  from  their  appearance  alone, 
to  regard  particular  colonies  on  gelatin  as  those  of  B.  coli  or 
its  varieties.  "Such  colonies,"  he  remarked  "could  not  without 
animal  experiment  be  declared  not  to  be  the  bacillus  of 
pseudo-tuberculosis.  Moreover  they  might  be  neither  B.  coli 
nor  its  varieties  nor  the  bacillus  of  pseudo-tuberculosis  but 
some  totally  different  organism." 

W.  B.  M.  Martin  (1911)  has  published  photographs  showing 
the  different  appearances  presented  by  colonies  of  the  gono- 
coccus  grown  from  the  same  strain  and  on  the  same  media. 

3.  In  the  third  place  it  is  known  that  the  addition  of 
various  substances  to  a  culture  medium  will  modify  the 
character  of  bacterial  colonies  growing  on  it.   Thus,  Penfold 
(1911  B,  c)  observed  that  the  addition  to  an  agar  medium  of 
certain  carbohydrates  developed  papillae  on  the  surface  of 
the  colonies  of  many  organisms.    B.  typhosus  exhibits  this 
papillae  formation  on  agar  containing  lactose,  dulcite,   or 


CH.  iv]         VARIATIONS  IN  MORPHOLOGY  49 

isodulcite.  He  found  that  on  raffinose  agar  B.  paratyphoid 
B  strains  produced  papillae  but  B.  Aertryck  strains  failed 
to  do  so,  and  that  this  difference  between  the  two  organisms 
was  sufficiently  constant  to  be  of  value  in  distinguishing 
between  them.  The  formation  of  papillae  indicated,  in  certain 
cases,  the  acquirement  on  the  part  of  some  members  of  the 
strain  of  power  to  ferment  the  carbohydrate  added  to  the 
medium. 

4.  Heating  a  strain   of  organisms  before  subculturing 
them  has  been  observed  to  modify  the  characters  of  the 
colonies  formed  (Bainbridge,  1903). 

5.  Finally,  as  a  result  of  passage,  the  type  of  colony 
formed  by  a  strain  of  bacteria  may  be  modified.   Thus  Adami, 
Abbott  and  Nicholson  (1899)  injected  into  a  rabbit  a  strain 
of  typical  B.  coli.  The  organism  recovered  formed  on  agar 
colonies  closely  resembling  those  of  S.  pyogenes.   By  intra- 
peritoneal  passage  through  three  guineapigs  typical  B.  coli 
were  obtained  once  more. 

VARIATON  IN  OTHER  MORPHOLOGICAL  CHARACTERS. 

Detailed  reference  has  not  been  made  to  variation  in 
other  morphological  characters,  such  as  motility,  pigment 
formation,  the  development  of  capsules  and  staining  properties, 
since  these  are  well  known  to  vary  greatly  at  different  times 
and  under  different  conditions.  A  few  examples  of  such 
variation  will  be  found  in  the  earlier  sections  (vide  Chap.  11). 


D. 


CHAPTER  V 

VARIATIONS  IN  FERMENTING  POWER 

THE  FERMENTATION  OF  CARBOHYDRATES. 

THE  process  of  fermentation  in  the  case  of  themono-saccharides 
or  glucoses — the  compounds  most  readily  fermented  by  the 
action  of  bacteria — consists  of  two  stages,  (i)  the  splitting  of 
the  "glucoses"  with  the  formation  of  acids  (formic  acid, 
lactic  acid)  and  (ii)  the  conversion  of  the  acid,  by  a  process 
of  hydration,  into  simpler  substances  most  of  them  gaseous 
(carbon  dioxide,  hydrogen,  methane,  etc.).  In  the  case  of  the 
di-saccharides  (lactose,  maltose,  saccharose)  an  earlier  stage 
must  first  be  completed,  namely  the  "inversion"  of  these 
substances  into  glucose.  This  preliminary  change  is  not 
easily  recognised,  but  the  two  final  stages  of  the  process  are 
sufficiently  indicated  by  the  formation,  respectively,  of  acid 
and  of  gas. 

The  power  possessed  by  certain  bacteria  to  bring  about 
the  fermentation  of  carbohydrate  compounds  is  subject  to 
variation  to  a  remarkable  degree.  Different  strains  of  the 
same  organism  may  differ  from  each  other  in  their  "  sugar  " 
reactions.  The  same  strain  may  vary  from  time  to  time 
during  cultivation,  apparently  spontaneously.  In  other  cases 
the  effect  can  be  traced  to  the  conditions  of  growth  and  is 
found  to  depend  upon  such  factors  as  the  temperature,  the 
presence  of  oxygen,  the  atmospheric  pressure,  the  age  of  the 
culture,  the  age  of  the  medium  and  its  composition.  The  power 
to  produce  fermentation  may  be  modified  by  symbiosis.  It 
is  sometimes  altered  in  the  case  of  carriers,  after  animal 
"passage"  and  in  the  course  of  a  disease.  New  fermenting 
properties  may  be  developed  SiS&remltofprolongedcultivation 
in  a  particular"  sugar,"  or  bya  process  of  "artificial  selection" 


CH.  v]    VARIATIONS  IN  FERMENTING  POWER         51 

I.  Different  strains  may  possess  different  fermenting 
properties. 

The  typical  pneumococcus  ferments  saccharose  (a  di- 
saccharide),  mannite  (a  polyatomic  alcohol)  and  inulin  (a 
starch).  Its  power  to  ferment  inulin  is  a  feature  upon 
which  reliance  is  placed  in  differentiating  the  organism  from 
other  members  of  the  streptococcus  group.  Eyre,  Leatham 
and  Wash  bourn  (1906)  found  that  out  of  14  different 
strains  examined  by  them  4  failed  to  ferment  inulin,  an  equal 
number  failed  to  ferment  mannite  and  3  failed  to  ferment 
saccharose. 

Strains  of  the  meningococcus  obtained  from  epidemic 
and  from  sporadic  cases  of  meningococcal  meningitis  exhibit 
differences  in  their  fermenting  properties  (Batten).  Arkwright 
(1909)  describes  several  strains  of  the  meningococcus  which 
failed  to  ferment  any  sugars — in  some  cases  "  permanently," 
and  in  other  cases  for  varying  periods  after  their  isolation. 

Wilson  (1908)  analyses  the  "sugar  reactions"  in  the  case 
of  44  gas  producing  coliform'  organisms  obtained  from  the 
urine  of  patients  suffering  from  cystitis  and  pyelitis.  The 
various  strains  showed  an  extraordinary  diversity  in  their 
fermenting  properties. 

Many  observers  have  recorded  marked  differences  in  fer- 
menting properties  between  different  strains  of  dysentery 
bacilli.  One  example  will  suffice.  Bahr  (1912)  collected  28 
different  strains  of  dysentery  bacilli  in  Fiji.  He  tested  the 
power  of  these  strains  to  ferment  six  sugars  (dextrose, 
dulcite,  maltose,  saccharose,  lactose  and  mannite)  with  the 
result  that  the  28  strains  formed  no  less  than  7  distinct 
groups.  The  addition  of  further  sugars  for  test  purposes 
would  no  doubt  have  revealed  still  more  varieties. 

Arkwright  (1909)  mentions  a  strain  of  gonococciis,  isolated 
from  the  urethral  discharge  in  a  case  of  acute  gonorrhoea, 
which  fermented  glucose  and  maltose  but  not  saccharose, 
thus  resembling  most  strains  of  the  meningococcus  and 
differing  from  the  typical  gonococcus  which  is  a  non-fermenter 
of  maltose.  W.  B.  M.  Martin  (1911)  describes  an  atypical 
strain  of  the  gonococcus,  isolated  in  pure  culture  from  the 

4—2 


52        VARIATIONS  IX  FERMENTING  POWER     [CH.  v 

knee  joint,  which  did  not  ferment  glucose,  levulose,  maltose 
or  saccharose. 

Gordon  (quoted  Martin,  1911)  when  testing  the  "sugar 
reactions"  of  25  strains  of  micrococcus  catarrhaUs,  discovered 
3  strains  which  fermented  glucose,  maltose,  galactose  and 
saccharose — none  of  which  sugars  are  normally  fermented  by 
this  organism. 

Strains  of  B.  diphtheriae  are  very  variable  in  their  action 
on  lactose  and  saccharose  (Graham  Smith,  1906). 

Klotz  (1906)  describes  a  coliform  organism,  quickly  ag- 
glutinated by  high  dilutions  of  typhoid  serum,  which  differed 
from  B.  typkosus  in  being  able  to  ferment  glucose,  lactose 
and  saccharose. 

Many  other  examples  might  be  given  of  differences  in 
fermentation  properties  displayed  by  different  strains  of  the 
same  organism. 

II.  The  same  strain  may  vary  spontaneously  during 
cultivation. 

Arkwright  (1909)  mentions  a  strain  of  the  meningococcus 
which  when  first  tested  fermented  no  sugars  :  subsequently, 
throughout  a  period  of  many  months,  it  fermented  maltose 
only  ;  finally,  after  10  months  artificial  culture,  it  fermented 
both  maltose  and  glucose.  Another  of  his  strains,  on  the 
other  hand,  at  first  fermented  both  maltose  and  glucose  but 
later  fermented  neither. 

Rosenow  (1914)  obtained  a  strain  of  haemolysing  strepto- 
cocci from  the  throat  in  a  case  of  Scarlet  fever.  A  culture 
on  blood  agar  yielded  two  distinct  kinds  of  colonies,  (a) 
colonies  of  a  haemolysing  organism  which  fermented  mannite 
but  failed  to  ferment  maltose  and  saccharose,  (b)  green 
producing  colonies  of  a  non-haemolysing  organism  which 
would  not  ferment  mannite  but  fermented  maltose  and 
saccharose.  The  two  strains  differed  also  in  their  patho- 
genicity. 

Andrewes  and  Gordon  (1905-6)  found  that i%  an  undoubtedly 
pure"  strain  of  staphylococcus pyogenes  aureus,  which  yielded 
a  brilliantly  pigmented  culture,  produced  on  subculture 


CH.  v]    VARIATIONS  IN  FERMENTING  POWER        63 

colonies  some  of  which  were  coloured  and  others  white.  In 
one  experiment  the  coloured  strain  formed  acid  in  salicin 
and  coniferin,  which  neither  the  original  strain  nor  the  white 
colonies  was  able  to  accomplish. 

Klotz  (1906)  has  described  a  coliform  organism  which  did 
not  ferment  lactose  or  saccharose  when  first  isolated  from 
water  but  fermented  both  after  48  hours'  growth  on  the  media. 

Horrocks  (1911)  describes  a  strain  of  B.  typhosus  which 
after  3  days'  growth  on  bile-salt-glucose-litmus-agar  gave 
typical  fermentation  tests  but  a  week  later  was  found  to  have 
acquired  the  power  to  ferment  lactose,  dulcite  and  salicin. 

Normally  B.  typhosus  ferments  glycerine.  Penfold  (1910  A) 
found  that  an  old  laboratory  culture  of  this  organism  on  agar, 
when  plated  out,  gave  some  colonies  which  fermented  glycerine 
and  others  which  failed  to  do  so  even  after  five  successive 
subcultures  had  been  made  into  peptone  water. 

Many  observers  have  recorded  instances  of  dysentery 
bacilli  acquiring  during  cultivation  the  power  of  fermenting 
sugars  which  previously  they  were  unable  to  attack — such  as 
(in  the  case  of  the  "Shiga"  organism)  mannite  (Torrey,  1905), 
the  di-saccharides  (Kruse,  quoted  by  Bahr).  Bahr  (1912) 
records  the  loss,  on  the  part  of  an  atypical  "Shiga"  organism, 
of  power  to  ferment  saccharose  after  6  months  subculture, 
and  maltose  after  7  months  subculture  ;  the  loss  on  the  part 
of  an  atypical  "Flexner"  organism  of  power  to  ferment 
lactose  after  4  months,  accompanied  by  a  temporary  loss  of 
the  power  to  ferment  maltose.  He  quotes  records  of  a  similar 
loss  of  power  to  ferment  lactose  after  4  years  (Morgan),  and 
maltose  after  9  years  (Lentz).  Another  strain  of  the  "Flexner" 
type  (after  a  month's  subculture)  produced  a  feebly  acid 
reaction  in  mannite  at  the  end  of  24  hours  but  after  10  days 
further  incubation  the  medium  became  definitely  alkaline. 
After  further  cultivation  for  some  weeks  it  produced  acidity 
in  mannite  in  24  hours.  Its  action  on  maltose  varied  greatly. 

Sorenson  (1912,  quoted  by  Dobell)  has  recorded  the  case 
of  a  patient,  suffering  from  glycosuria  who  also  developed 
pneumaturia.  The  gas  formation  was  found  to  be  due  to  a 
peculiar  bacillus,  " B.  pneumaturiae"  which  had  gained 


54        VARIATIONS  IN  FERMENTING  POWER    [CH.  v 

access  to  the  bladder.  This  organism  was  isolated  and  on 
cultivation  was  found  to  ferment  glucose,  lactose  and  sac- 
charose with  the  production  of  much  gas.  After  two  years 
the  patient  recovered  spontaneously  from  the  pneumaturia, 
though  the  organism  was  discovered  still  to  be  present  in  the 
bladder.  Cultures,  however,  failed  to  yield  gas  on  sugar 
media.  About  a  year  later  the  strain,  which  had  been  sub- 
cultured  throughout  this  interval,  suddenly  re-acquired  the 
property  of  producing  gas,  and  "shortly  after"  the  patient 
commenced  to  suffer  again  from  pneumaturia. 

The  clock-like  precision  with  which  these  two  strains,  one 
on  artificial  media  and  the  other  in  the  human  body,  are 
stated  to  have  exhibited  this  spontaneous  variation,  after  the 
same  interval  of  time  and  in  spite  of  the  difference  in  their 
respective  environment,  may  perhaps  excuse  some  incredulity. 
One  suspects  that  further  investigation  would  have  shown 
the  modification  to  exist  in  the  sugar  media  employed  rather 
than  in  the  bacteria. 

III.  Fermenting  properties  may  be  modified  by  the 
conditions  of  growth. 

1.  The  influence  of  temperature.  Wilson  (1910)  isolated 
B.  typhosus  from  the  urine  of  a  "  carrier."  At  a  temperature 
of  22°  C.  this  organism  fermented  lactose  litmus-agar  in  2  days 
but  at  a  temperature  of  37°  C.  no  acidity  was  produced  after 
a  month's  incubation.  The  absence  of  acidity  at  the  higher 
temperature  might  be  accounted  for  on  the  supposition  that 
the  products  of  the  proteid  decomposition,  at  this  temperature, 
neutralised  the  acid  formed  during  the  process  of  fermen- 
tation. Wilson  proved  that  this  was  not  the  true  explanation 
by  estimating  the  amount  of  lactose  present  and  showing 
that  it  had  not  been  attacked. 

Coplans  (1909)  mentions  some  strains  of  B.  coli  which 
showed  the  reverse  phenomenon,  fermenting  dulcite  more 
readily  at  37°  than  at  20°  C. 

Adami  has  described  an  alteration  in  the  fermenting  pro- 
perties of  B.  coli  communis  after  subjection  to  a  high  tem- 
perature in  the  presence  of  peritoneal  fluid. 


CH.  v]    VARIATIONS  IN  FERMENTING  POWER        55 

2.  The  influence  of  oxygen.  Torrey  (1905)  found  that 
the  power  of  a  certain  dysentery  bacillus  to  ferment  maltose 
was  augmented  by  alternate  aerobic  and  anaerobic  culture. 

Wilson  describes  an  atypical  B.  typhosus  which  slowly 
fermented  lactose  in  a  litmus-broth  tube  but  produced 
fermentation  in  3  days  when  the  same  medium  was  poured 
into  a  Petri  dish. 

Andre wes  and  Horder  (1906)  mention  a  streptococcal 
strain  which  failed  to  ferment  lactose  under  ordinary  con- 
ditions but  did  so  readily  when  grown  anaerobically. 

3.  The  influence  of  atmospheric  pressure .  Harden  (1901) 
has  shown  that  the  amount  of  formic  acid  produced  by  B. 
coli  from  glucose,  at  the  ordinary  pressure  of  the  atmosphere, 
is  very  small.   Under  greater  pressure  the  yield  of  acid  is 
increased,  while  at  the  same  time  the  amount  of  gas  formed 
is  diminished.   In  other  words,  the  final  stage  in  the  process 
of  fermentation,  which  consists  in  the  conversion  of  the  acid 
into  various  gases,  is  inhibited. 

4.  The  age  of  the  culture.  Older  cultures  of  B.  diphtheriae 
usually  ferment  both  glycerine  and  lactose  ;  a  young  culture 
of  the  same  organism  can  attack  neither  of  these  substances 
(Muir  and  Ritchie). 

5.  The  age  of  the  medium.  The  streptococcus  pyogenes 
normally  does  not  ferment  saccharose,  raffinose  or  salicin, 
but  if  old  media  be  used  this  organism  will  ferment  both  the 
first  two  substances,  and  even  the  last  named  in  the  course  of 
a  week  (Martin).   On  the  other  hand  B.  diphtheriae  which 
gives  its  characteristic  "  sugar  reactions  "  on  fresh  beef  serum, 
fails  to  do  so  if  this  medium  is  old  (Fisher,  1909). 

6.  The   composition  of  the  medium.    The  addition  of 
carbolic  acid  in  small  quantities  to  the  media  used  destroys 
the  natural  fermenting  properties  of  many  bacteria  (Penfold, 
1911  B). 

The  presence  of  sodium  benzoate  inhibits  the  power  of 
B.  coli  to  produce  gas  from  dextrose,  one  of  the  most  stable 
and  fundamental  differences  separating  the  coli  from  the 
typhoid-dysentery  group  (Herter,  1909).  Penfold  (1911  c) 
found  that  many  intestinal  organisms  (B.  coli,  B.  enteritidis 


56        VARIATIONS  IN  FERMENTING  POWER    [CH.  v 

Gaertner,  B.  paratyphosus  B,  etc. )  by  growth  on  monochlor- 
acetic-acid-agar,  were  deprived  of  their  power  to  form  gas 
from  glucose  and  other  sugars ;  they  retained,  however,  their 
power  to  ferment  the  corresponding  alcohols.  He  found  that 
the  variation  in  character  was  maintained,  even  on  daily 
subculture,  for  many  "  generations." 

7.  The  source  of  a  strain,  that  is  to  say  the  nature  of  the 
medium  from  which  it  is  isolated,  may  determine  certain 
variations  in  fermenting  power.  Thus,  Revis  (1908)  found  that 
strains  of  B.  coli  cultivated  from  milk  were  frequently  able 
to  ferment  saccharose.  Moreover  a  strain  obtained  from 
cow  dung,  which  was  unable  to  ferment  saccharose,  acquired 
the  power  to  do  so  after  being  grown  in  milk.  Wilson  (1909) 
found  that  many  coliform  organisms  isolated  from  urine  had 
lost  the  property  of  forming  gas  from  dextrose.  Adami, 
Abbott  and  Nicholson  (1899)  describe  strains  of  B.  coli  grown 
in  ascitic  fluid  as  being  unable  to  ferment  glucose  or  dextrose 
broth — this  power  being  only  partially  regained  after  three 
passages  through  the  guineapig. 

IV.  Symbiosis  may  influence  fermenting  power. 

Major  Horrocks's  experiments  (1911)  are  of  interest  in 
this  connection.  He  found  in  two  experiments  that  a  typical 
strain  of  B.  typhosus  lost  its  power  to  ferment  "  sugars  "  when 
grown  in  the  presence  of  a  strain  of  B.  coli  derived  from 
the  urine  of  a  "typhoid  carrier."  In  one  case  reversion  in 
character  took  place  on  further  subculture.  He  found,  in 
a  third  experiment,  that  a  strain  of  B.  typhosus  derived 
from  the  urine  of  a  "  carrier,"  lost  both  its  fermenting  and 
its  agglutinating  properties  when  grown  in  the  diluted,  filtered 
urine  of  another  "typhoid  carrier." 

V.  Variation  in  fermenting  power  is  found  in  organisms 
isolated  from  "  carriers." 

The  strain  of  B.  typhosus  isolated  by  Wilson  from  the 
urine  of  a  "  typhoid  carrier ''  was  found  to  have  acquired  the 
power  to  ferment  lactose  and  saccharose,  and  the  power  also 
to  produce  "much  gas"  from  mannite  and  maltose. 


CH.  v]    VARIATIONS  IN  FERMENTING  POWER        57 

VI.  Fermenting  power  may  be  altered  by  "animal 
passage" 

Klotz  (1906)  isolated  from  water  an  atypical  organism  of 
the  B.  coll  group.  This  organism,  after  a  residence  of  144 
days'  duration  in  a  celluloid  sac  within  the  peritoneal  cavity 
of  a  rabbit,  showed  a  temporary  loss  of  power  to  ferment 
glucose,  saccharose  and  lactose.  The  loss  was  most  marked 
in  the  case  of  lactose  which  was,  however,  again  fermented 
in  the  4th  subculture  into  lactose  broth,  and  also  by  the  8th 
subculture  on  ordinary  media  (agar). 

'Peckham  (1897)  introduced  B.  coli  into  the  peritoneal 
cavity  in  sufficient  numbers  to  set  up  a  fatal  inflammatory 
process.  The  organism,  recovered  on  the  death  of  the  animal 
4  days  later,  showed  slight  changes  in  fermenting  power. 

Horrocks  (1911)  describes  a  strain  of  B.  typhosus  which, 
in  the  course  of  cultivation,  acquired  the  power  to  ferment 
lactose,  dulcite  and  salicin.  "  Passage  "  through  4  guineapigs 
destroyed  the  power  to  ferment  lactose  and  dulcite  but  after 
4  further  passages  the  power  was  regained. 

Bahr  (1912)  describes  experiments  in  which  flies  were  fed 
on  dysentery  bacilli  (both  of  the  "Shiga"  and  of  the 
"  Flexner  "  type)  and  states  that  the  organism  recovered  from 
the  intestinal  tract  in  several  cases,  "  undoubtedly  derived 
from  the  bacillus  originally  fed  to  the  flies,"  gave  different 
sugar  reactions.  The  sugar  reactions  had,  for  9  months 
previously,  remained  constant  on  repeated  trials.  One  "  Shiga  " 
organism  had  acquired  the  power  to  ferment  maltose.  In  the 
case  of  a  "Flexner"  organism,  the  power  of  fermenting 
mannite  (upon  which  the  distinction  between  the  acid  and 
non-acid  types  depends)  was  diminished,  fermentation  only 
occurring  after  4  days'  incubation.  Other  organisms  of  the 
"Flexner"  type  had  acquired  the  power  to  ferment  maltose 
and  saccharose.  Both  types  of  organism  on  subculture  re- 
verted to  their  original  characters  in  the  course  of  several 
months. 

Adami,  Abbott  and  Nicholson  (1899)  obtained  from  human 
ascitic  fluid  an  atypical  B.  coli  which  had  completely  lost  its 
fermenting  power.  This  power  was  restored  after  a  series 


58        VARIATIONS  IN  FERMENTING  POWER    [CH.  v 

of  three  intra-peritoneal  passages  through  the  guineapig. 
Another  similar  strain  from  ascitic  fluid  after  several  passages 
was  still  unable  to  produce  gas  from  glucose  or  dextrose. 

VII.  Variation  in  the  fermenting  power  of  organisms 
may  arise  during  the  course  of  a  disease. 

Connal  (1910)  has  shown  that  in  the  later,  chronic,  stages 
of  cerebrospinal  fever  the  meningococcus  isolated  from  the 
spinal  fluid  has  lost  its  power  to  ferment  dextrose. 

Adami,  Abbott  and  Nicholson  (1899)  in  describing  diplo- 
coccic  forms  of  B.  coli,  isolated  from  the  ascitic  fluid  in 
cases  of  hepatic  cirrhosis,  mentions  that  they  had  lost  the 
power  of  fermenting  glucose  and  lactose  broths. 

VIII.  The  power  to  ferment  a  "sugar"  may  be  acquired 
by  bacteria  after  prolonged  growth  in  a  medium  containing 
that  sugar. 

Hiss  (1904)  has  described  a  bacillus  of  the  dysentery  group 
which  acquired  the  power  to  ferment  maltose  after  being 
grown  for  some  time  in  a  maltose  medium. 

Twort  (1907)  found  that  dysentery  bacilli  (both  the 
"Flexner"  and  the  "Shiga"  type)  which  did  not  normally 
ferment  saccharose,  did  so  after  cultivation  in  a  medium 
containing  this  ingredient,  and  by  similar  means  the  true 
"  Shiga  Kruse  "  organism  was  induced  to  ferment  lactose. 

In  the  same  way  he  found  that  members  of  the  paratyphoid 
group,  after  prolonged  cultivation  on  a  saccharose  medium, 
all  acquired  the  power  to  ferment  it,  while  a  strain  of  B. 
typhosus  acquired  the  power  to  ferment  lactose,  but  only 
after  two  years'  "training."  The  same  organism  could  be 
made  to  ferment  dulcite  in  a  very  shorter  period,  2  or  3  weeks 
being  sufficient. 

Penfold  (1910)  trained  B.  typhosus  to  ferment  dulcite  in 
a  period  of  10  days,  arabinose  in  2  or  3  months  and  isodulcite 
after  varying  intervals.  He  failed  however  to  develop  new 
fermenting  powers  on  the  part  of  the  same  organism  towards 
lactose  after  a  15  months'  trial,  or  towards  saccharose  and 
other  substances  after  9  months. 


CH.  v]    VARIATIONS  IN  FERMENTING  POWER        59 

Burton  Bradley  (1910)  repeated  these  experiments  with 
success  as  regards  dulcitol  and  arabinose. 

Penfold  states  that  in  the  case  of  B.  typhosus  the  acquire- 
ment of  new  fermenting  properties  on  the  part  of  certain 
individuals  of  the  strain  is  indicated,  in  some  instances,  by 
the  formation  of  papillae  on  the  colonies.  He  observed  this 
to  take  place  in  a  medium  containing  dulcite  or  sorbite.  In 
his  opinion  considerable  permanency  in  the  new  fermenting 
power  was  indicated  if  the  papillae  formation  arose  early  and 
without  subculture. 

IX.  Variation  in  fermenting  power  may  be  brought 
about  by  a  process  of  Artificial  Selection. 

Goodman  (1908)  made  a  series  of  cultures  of  B.  diph- 
theriae  in  dextrose  broth.  From  this  series  he  selected  the 
tube  giving  the  greatest  acidity,  when  titrated  against  a 
standardised  soda  solution,  and  the  tube  giving  the  lowest 
acidity.  From  each  of  these  two  tubes  he  made  a  fresh  series 
of  cultures  and,  after  3  days'  growth  at  37°  C.,  he  chose  out  of 
the  more  acid  series  the  tube  giving  the  greatest  acidity,  and 
out  of  the  less  acid  series  the  tube  giving  the  lowest  acidity. 
With  these  two  tubes  he  made  a  fresh  double  series  of 
cultures  and  repeated  the  process  of  selection.  After  repeating 
this  36  times  he  obtained  one  culture  which  produced  intense 
acidity  in  dextrose,  and  a  second  culture  which  failed  to  pro- 
duce acidity  in  dextrose  at  all  and  in  fact  made  it  more 
alkaline.  These  two  strains  were  then  tested  with  other 
sugars  and  it  was  found  that,  in  both  cases,  the  power  to 
ferment  maltose  was  diminished  while  the  power  to  ferment 
saccharose  was  increased.  Their  action  on  dextrin  was  not 
affected. 

It  is  to  be  noted  that  the  difference  in  fermenting  power 
between  these  two  selected  strains  was  as  great  as  that 
normally  existing  between  B.  diphtheriae  -and  B.  pseudo- 
diphtheriae. 

This  process  of  continued  selection  in  opposite  directions 
does  not  necessarily  succeed  in  developing  strains  of  extreme 
type.  For  example,  Buchanan  and  Traux  (quoted  by  Rettger 


60        VARIATIONS  IN  FERMENTING  POWER    [CH.  v 

and  Sherrick,  1911)  failed  to  develop  high  and  low  acid 
forming  strains  of  streptococcus  lacticus  and  Glenn  (1911) 
records  a  similar  failure  in  the  case  of  B.  proteus. 

THE  SIGNIFICANCE  OF  VARIATIONS  IN  THE 
"SUGAR  REACTIONS." 

A  consideration  of  the  action  of  bacteria  on  carbohydrates 
and  a  comparison  with  the  similar  action  of  other  (vegetable) 
cells,  such  as  yeast,  justify  certain  conclusions. 

1.  The  splitting  up  of  the  carbohydrate  is  undoubtedly 
effected    through    the  agency    of   enzymes  or   "  organised 
ferments,"  the  functions  of  which  are  inhibited  or  destroyed 
by    antiseptics,    such    as    carbolic    acid,    sodium    beuzoate, 
monochlor-acetic  acid. 

2.  The    fermentation  of  a  particular  carbohydrate  is 
dependent  on  the  activity  of  a  particular  ferment,  so  that  the 
power  to  ferment  one  carbohydrate  is  quite  independent  of 
the  power  to  ferment  another. 

This  is  well  illustrated  by  Goodman's  experiments.  The 
two  strains  obtained  by  him  from  a  culture  of  B.  diphtheriae, 
one  with  greatly  increased  fermenting  power  towards  dextrose 
and  the  other  with  almost  complete  absence  of  such  power, 
both  showed  an  augmentation  of  their  power  to  ferment 
saccharose  and  a  diminution  of  their  power  to  ferment 
maltose.  Penfold  succeeded  in  modifying  strains  of  B.  coli, 
B.  enteritidis  Gaertner  and  B.  Grilnthal  by  growing  them 
in  the  presence  of  a  certain  antiseptic,  with  the  result  that 
they  lost  the  power  to  produce  gas  from  the  sugars  while 
still  retaining  the  power  to  produce  gas  from  the  corresponding 
alcohols. 

3.  The    three    stages    in   the   process    of   fermentation, 
namely  the  preliminary  stage  of  "  inversion  "  (in  the  case  of 
the  di-saccharides)  and  the  two  final  stages  in  which  acid  is 
first  formed  and  then  split  up  into  gases,  are  due  to  the 
activity  of  three  different  enzymes.   Failure  to  produce  gas 
may  be  due  to  the  absence  or  the  inhibition  of  any  one  of 
these  three  enzymes.   Failure  to  produce  acidity  may  be  due 


CH.  v]    VARIATIONS  IN  FERMENTING  POWER        61 

to  the  absence  or  inhibition  of  either  the  "  inverting  "  ferment 
or  the  "  acid-forming  "  ferment. 

That  the  several  steps  in  the  process  of  fermentation 
result  from  the  activity  of  different  enzymes  is  suggested  by 
the  following  considerations.  Typhoid  and  dysentery  bacilli 
never,  of  themselves,  produce  gas  and  cannot  be  made  to  do 
so  by  training  or  selection  ;  both  however  produce  acidity  in 
dextrose  and  mannite  and  can  be  "trained"  to  do  so  in 
lactose. 

B.  coli  is  known  to  produce  formic  acid  from  glucose  and 
then  to  split  up  the  formic  acid  into  gases ;  Penfold  has 
shown  that  growth  on  mpnochlor-acetic-acid-agar  may  inter- 
fere with  the  "formic  acid-forming"  property  of  this  organism 
without  affecting  its  "  formic  acid-splitting  "  power,  that  is  to 
say  its  power  to  form  gas  from  formic  acid. 

4.  This  observer  has  gone  still  further  and  has  shown  that, 
during  the  stage  of  add  production,  more  than  one  kind  of 
acid  may  be  formed  by  an  organism  but  that  the  formation 
of  each  acid  is  the  work  of  a  special  enzyme.   The  inhibition 
of  the  "  formic  acid-forming  "  enzyme  in  the  case  of  B.  coli 
did  not  interfere  with  the  production  of  acidity  in  dextrose. 

5.  Penfold  likewise  showed  that  the  splitting  up  of  each 
acid,  with  the  formation  of  gases,  was  the  work  of  a  special 
enzyme  and  that  an  enzyme  which  could  produce  gas  from  one 
acid  could  not  do  so  from  another.  The  strain  of  B.  coli  which 
was  deprived  of  its  power  to  form  formic  acid  was,  on  this 
account,  deprived  of  its  power  to  produce  gas,  for,  although  the 
organism  could  produce  other  acids  (as  shown  by  the  reaction 
of  the  medium)  it  could  not  split  these  up.   If  however  sodium 
formate  was  added  to  the  medium  the  organism  at  once  yielded 
gas ;   or,   again,   if  the  organism  were  grown  in   dextrose 
with  B.  typhosus  (which  possesses  the  power  of  producing 
formic  acid  from  dextrose  but  cannot  split  the  acid  up)  it 
once  more  yielded  gas.   It  is  obvious,  then,  that  in  the  case  of 
B.  coli  its  "  acid-splitting  "  enzyme  is  only  capable  of  splitting 
up  formic  acid  and  cannot  form  gas  from  other  acids. 

6.  The  development  by  a  strain  of  bacteria  in  contact 
with  a  certain  sugar,  of  the  power  to  ferment  that  sugar  is  an 


62        VARIATIONS  IN  FERMENTING  POWER    [OH.  v 

example  of  adaptation  to  environment.  If  a  slow  fermenter 
of  dulcite  is  grown  in  a  medium  containing  some  other  sugar, 
such  as  dextrose,  its  power  to  ferment  dulcite  is  not  increased. 

7.  The  ability  to  split  up  the  sugar  is  apparently  an 
advantage  to  the  organism  concerned.   That  this  is  actually 
the  case  is  confirmed  by  the  observation  of  Penfold  that  the 
appearance  of  acidity  coincides  with  a  very  rapid  and  a  very 
marked  increase  in  the  number  of  organisms  present.   So 
constant  did  he  find  this  association  of  events  that  he  regards 
a  count  of  the  organisms  as  sufficient  by  itself  to  indicate  the 
occurrence  of  the  variation.    He  found,  moreover,  that  the 
addition  of  dulcite  to  peptone  water  containing  a  dulcite- 
fermenting  strain  of  B.  typhosus,  rendered  the  medium  capable 
of  supporting  a  population  many  times  greater  than  it  was 
able  to  support  alone.  The  addition  of  other  sugars  which  the 
organisms  could  not  ferment  did  not  lead  to  any  increase  in 
their  numbers. 

The  ability  to  ferment  the  sugar,  even  if  in  some  cases  it 
were  not  of  actual  benefit  to  the  fermenting  organism,  might 
still  prove  of  advantage  to  it  indirectly.  Marked  acidity  ot 
the  medium  is  known  to  be  unfavourable,  as  a  rule,  to 
bacterial  growth  ;  but  it  might  be  expected  that  the  acid 
producing  individuals  in  a  strain  would  be  unusually  resistant 
to  the  products  of  their  own  activity  and  that  their  growth 
would,  on  this  account,  be  inhibited  to  a  less  degree  than 
that  of  the  non-fermenters. 

8.  It  is  easy  to  understand  how  natural  selection  will 
cause  any  character  to  predominate  which  gives  the  possessors 
of  it  an  advantage  over  their  fellows.   This  is  the  obvious 
explanation  of  the  development  by  a  strain  of  bacteria,  when 
grown  on  a  certain  sugar,  of  the  power  to  ferment  that  sugar. 

Two  phases  of  the  phenomenon,  however,  call  for  further 
explanation,  namely  the  prolonged  incubation  period  and  the 
shortening  of  this  period  by  subculture. 

9.  The    incubation  period  may   be    explained   in   one 
of  three  ways. 

In  the  first  place  it  may  be  regarded  as  a  "  latent  period  " 
during  which  changes  occur  in  the  organisms  as  a  result  of 


CH.  v]     VARIATIONS  IN  FERMENTING  POWER        63 

their  contact  with  the  new  sugar,  such  changes  being  pre- 
paratory to  the  acquisition  on  their  part  of  new  fermenting 
powers.  The  observation  already  referred  to,  that  a  very 
rapid  increase  in  the  number  of  fermenting  organisms  occurs 
simultaneously  with  the  appearance  of  acidity,  tends  to 
support  this  hypothesis.  Penfold  has,  however,  disproved  it 
by  experiment.  He  observed  that  B.  typhosus  when  grown 
in  dulcite  broth  gradually  acquired  the  power  to  ferment  the 
sugar.  After  several  days  had  passed,  plating  out  on  dulcite 
agar  showed  that  95  per  cent,  of  the  strain  were  dulcite  fer- 
menters.  He  found  that  subcultures  from  the  non-fermenting 
colonies  into  dulcite  broth  took  the  same  length  of  time  as  the 
original  stock  to  produce  fermentation,  thus  showing  that 
they  had  not  undergone  any  preparatory  change  during  the 
first  incubation  period. 

In  the  second  place,  if  the  development  of  the  new  fer- 
menting power  is  dependent  in  the  first  instance  upon  the 
occurrence  of  a  spontaneous  "fluctuating"  variation  in  the 
required  direction,  and  such  variations  are  infrequent,  an 
interval  of  uncertain  length  must  necessarily  intervene  be- 
tween the  commencement  of  the  experiment  and  the  appear- 
ance of  the  variant  which  is  to  give  rise  to  the  new  strain. 
The  fact  that  the  length  of  the  incubation  period,  whenever 
a  certain  organism  is  "  trained  "  to  ferment  a  certain  sugar,  is 
fairly  constant,  disposes  of  this  argument. 

In  the  third  place,  the  original  non-fermenting  strain  might 
contain  a  very  few  fermenting  individuals,  in  insufficient 
numbers,  however,  to  give  any  evidence  of  their  presence. 
These  few  "fermenters"  would  possess  an  advantage  over  the 
"non-fermenters"  and  multiplying  more  rapidly  would,  in  time, 
outnumber  the  latter,  but  a  certain  period  would  necessarily 
elapse  before  they  gained  the  ascendancy.  Inasmuch,  however, 
as  the  original  strain  can  be  made  in  the  same  way  to  ferment 
a  number  of  different  sugars,  the  strain  must,  on  this  hypo- 
thesis, contain  at  one  and  the  same  time  fermenters  of  each  of 
these  different  sugars.  Even  if  this  be  granted  the  hypothesis 
offers  no  explanation  of  the  fact  (illustrated  by  the  behaviour 
of  B.  typhosus  in  dulcite  and  in  lactose  respectively)  that  in  the 


64        VARIATIONS  IN  FERMENTING  POWER    [CH.  v 

presence  of  one  sugar  the  fermenters  of  that  sugar  invariably 
gain  complete  ascendancy  in  the  course  of  a  few  days,  while  in 
the  presence  of  another  sugar  the  fermenters  of  it  invariably 
require  many  months  to  do  so. 

1 0.  The  shortening  of  the  incubation  period  on  subculture 
is  more  easily  explained.   When  a  few  bacteria  are  inoculated 
into  a  tube  of  broth  they  multiply  with  amazing  rapidity. 
There  is  a  limit,  however,  to  the  number  of  organisms  a  certain 
volume  of  the  medium  will  support — owing  not  only  to  the 
using  up  of  the  food  but  also  to  the  accumulation  of  waste 
products — so  that  after  a  time  multiplication  takes  place  much 
more  slowly.    Subculture  into  fresh  medium  gives  a  new 
impetus  to  reproduction.   The  "fermenters"  in  a  mixed  strain 
gain  the  ascendancy  by  virtue  of  their  capacity  to  utilise  the 
sugar,  which  enables  them  to  multiply  more  rapidly  than  the 
"non-fermenters."     Any  factor  therefore  which  accelerates 
the  rate  of  increase  of  both,  hastens  the  ultimate  mastery  of 
the  more  rapidly  multiplying,  that  is  to  say,  the  "fermenters.'* 

11.  "Artificial  selection"  appears  to  be  an  even  more 
powerful  factor  in  developing  a  particular  fermenting  power 
than  "natural  selection."    For  example,  Goodman  obtained 
by  artificial  selection  a  strain  of  B.  diphtheriae  which  had 
practically  lost  its  power  to  ferment  dextrose,  although  it 
had  been  subcultured  from  one  dextrose  media  to  another 
repeatedly  over  a  long  period. 

12.  When  an  organism  has  been  deprived  by  passage  or 
by  other  means,  of  its  power  to  ferment  a  particular  sugar, 
"reversion"  in   character  occurs  subsequently  on  ordinary 
media,  that  is  to  say  in  the  absence  of  the  particular  sugar  in 
question.    (The  addition  of  the  latter  hastens  "reversion,"  a& 
Klotz  has  shown,  but  its  presence  plays  only  a  subordinate 
part  in  the  process).    The  sequence  of  events  in  such  cases 
suggests  that  the  enzyme  is  temporarily  inhibited  in  its  action 
and  not  destroyed. 

13.  The  sudden  acquisition  on  the  part  of  a  strain  of  bac- 
teria of  power  to  ferment  a  certain  sugar  with  which  it  has  not 
been  in  contact,  is  more  difficult  to  explain — particularly  so 
when  such  a  variation  occurs  after  long  periods,  even  years,  of 


CH.  v]     VARIATIONS  IN  FERMENTING  POWER        65 

cultivation  in  one  medium,  during  which  repeated  examina- 
tions revealed  no  change  in  fermenting  properties. 

The  possibility  must  always  be  considered  that  the  strain 
may  have  been  subcultured  into  fresh  media  in  which  the  new 
sugar  was  accidentally  present  as  an  unrecognised  impurity, 
and  that  the  bacteria  "learnt"  to  ferment  this  impurity  after 
a  preliminary  "training."  They  would  be  more  likely  to  do 
this  if  the  process  of  subculturing  were  only  carried  out  at 
long  intervals  (as  might  easily  happen  in  the  case  of  a  stock 
culture)  for  this  would  afford  time  for  the  bacteria  to  exhaust 
the  normal  sugar  of  the  medium  and  their  survival  would  then 
depend  upon  their  power  to  utilise  traces  of  any  other  sugar 
that  might  be  present. 

The  possibility  also  suggests  itself  that  in  a  medium  con- 
taining a  di-saccharide  (lactose,  maltose,  saccharose)  inversion 
might  occur  to  a  slight  extent,  with  the  formation  of  traces  of 
a  simpler  mono-saccharide  (dextrose,  galactose)  which  the 
bacteria  growing  in  the  medium  would  then,  in  the  same  way, 
"learn"  to  ferment. 

Yet  a  third  possibility  is  that  the  particular  specimen  of 
the  "sugar"  used  to  test  the  fermenting  properties  of  a  strain 
of  bacteria  may  not  be  pure.  This  possible  source  of  error  is, 
however,  more  easily  guarded  against. 

Finally — even  if  it  be  admitted  that  a  variation  in  ferment- 
ing power  represents  an  adaptation  to  different  foodstuffs — we 
are  forced  to  conclude  that  different  members  of  a  strain  differ 
from  one  another  in  their  powers  of  adaptation ;  for,  when  an 
apparently  spontaneous  variation  in  fermenting  power  occurs 
during  cultivation,  in  a  strain  derived  originally  from  a  single 
bacterium,  fermenting  and  non-fermenting  individuals  may 
be  found  side  by  side  on  the  medium.  This  variation  between 
the  organisms  of  one  and  the  same  strain  we  are  quite  unable 
to  explain. 

THE  VALUE  OF  THE  SUGAR  REACTIONS. 

The  unsatisfactory  nature  of  the  "sugar  reactions,"  both 
as  a  means  of  identification  and  as  a  basis  for  classification, 
will  be  apparent  from  the  following  considerations. 

D.  5 


66        VARIATIONS  IN  FERMENTING  POWER     [CH.  v 

1.  In  the  first  place,  there  is  the  question  of  the  time 
allowance  to  be  made.  When  a  particular  organism  only 
produces  acidity  in  a  certain  sugar  at  the  end  of  an  incubation 
period  lasting  several  days,  one  is  in  doubt  whether  to  regard 
the  organism  in  question  as  a  slow  fermenter  of  that  sugar  or 
as  a  non-fermenter  of  it  which  has  acquired  a  new  character 
as  the  result  of  "training." 

2.  Secondly,  many  conditions,  as  we  have  shown,  modify 
the  normal  sugar  reactions  and  may  lead  to  erroneous  con- 
clusions.    A  strain  of  bacteria  may  ferment  a  sugar  at  a 
temperature  of  22°  C.  and  fail  to  do  so  at  37°  C. ;   an  old 
culture  may  ferment  substances  which  a  young  culture  is 
unable  to  do,  and  so  on. 

3.  In  the  third  place,  the  composition  of  the  medium  may 
be  responsible  for  conflicting  results.    It  is  almost  impossible 
to  obtain  many  of  the  carbohydrates  in  a  pure  form,  and  yet 
these  are  used  and  conclusions  are  based  on  the  reactions  they 
give.    Others  can  be  obtained  pure  but  are  then  too  costly 
for  general  use  and  the  commercial  "sugar"  is  substituted. 
Different  specimens  of  the  same  carbohydrate,  even  when 
reasonably  pure,  msty  give  different  results.    This  is  the  case 
with  the  starch  inulin,  the  fermentation  of  which  is  an  import- 
ant distinction  between  the  pneumococcus  and  other  members 
of  the  streptococcus  group.    The  process  of  sterilisation  is  a 
difficult  one.   If  subjected  to  too  high  a  temperature,  particu- 
larly in  the  presence  of  alkaline  material,  the  sugar  may 
undergo  a  change  in  composition.    If  the  temperature  is  not 
raised  sufficiently  sterilisation  may  be  incomplete.     If  the 
vessels  holding  the  medium  are  not  made  of  the  best  Jena  glass, 
there  is  a  danger  of  the  glass  yielding  a  considerable  amount 
of  alkali  to  the  medium  during  sterilisation  (W.  B.  M.  Martin, 
1911).    If  the  various  media  are  kept  for  any  length  of  time 
before  use,  the  carbohydrates  may  deteriorate  and  lead  to 
apparently  abnormal  sugar  reactions.    Examples  of  this  have 
already  been  given.   Finally,  if  the  medium  is  very  alkaline  in 
the  first  instance,  or  if  it  is  rendered  so  by  the  decomposition 
of  the  peptone  present,  the  acid  reaction  may  be  masked. 

4.  Even  if  the  composition  of  the  medium  is  beyond  re- 


CH.  v]    VARIATIONS  IN  FERMENTING  POWER        67 

proach,  the  reactions  are  not  necessarily  constant.  There  is 
a  great  deal  of  evidence  to  show  that  spontaneous  variations 
frequently  occur. 

5.  In  any  case,  the  classification  of  bacteria  according  to 
their  action  on  certain  carbohydrates  is  a  very  artificial  one. 
Twort  emphasises  the  fact  that  the  difference  between  one 
organism  which  produces  such  slight  acidity  that  the  alkaline 
reaction  of  the  medium  completely  masks  it,  and  another 
organism  which  produces  a  slight  but  definite  acid  reaction, 
is  no  greater  than  the  difference  between  the  latter  organism 
and  a  third  which  produces  marked  acidity.   In  the  same  way 
the  difference  between  an  organism  which  yields  acidity  in 
24  hours  and  one  which  requires  48  or  even  72  hours  to  do  so, 
cannot  be  regarded  as  a  fundamental  one.   It  is  merely  a  matter 
of  degree. 

6.  Further,  the  decision  as  to  which  group  of  carbohydrate 
compounds  (the  sugars,  the  glucosides,  the  starches,  etc.)  shall 
constitute  the  test  substances  is  a  purely  arbitrary  one, 
depending  not  infrequently  upon  their  cheapness  and  the 
facility  with  which  they  can  be  obtained.    If  one  group  of 
carbohydrates  be  chosen  a  certain  classification  will  follow ;  if 
another  group  be  selected  an  entirely  different  classification 
may  result. 

The  only  justification  for  founding  a  classification  upon 
one  series  of  experiments  rather  than  upon  the  other  is  the 
fact  that  the  classification  so  obtained  corresponds  more  closely 
to  differences  brought  out  in  other  ways,  such  as  differences 
in  agglutination  or  pathogenicity. 

If  these  other  differences  are  inconstant  and  distinctions 
based  on  them  have  been  found  to  be  unreliable,  then  a  series 
of  fermentation  tests  designed  to  correspond  with  them  is  at 
once  suspect  and  cannot  be  trusted  as  a  final  appeal.  If  on 
the  other  hand  these  other  differences  (in  agglutination,  patho- 
genesis,  etc.)  are  constant  and  have  been  found  to  justify  a 
certain  division  into  "species,"  a  series  of  fermentation  tests 
which  correspond  to  them  may  afford  a  very  much  simpler 
method  of  deciding  to  which  of  these  species  a  certain 
organism  belongs,  and  also  of  separating  one  species  from 

5—2 


68        VARIATIONS  IN  FERMENTING  POWER     [OH.  v 

another  by  "plating  out"  a  mixed  culture  on  the  appropriate 
medium. 

It  is  this  that  constitutes  the  real  value  of  the  "sugar 
reactions."  In  other  words  the  tests  are  of  more  use  for 
purposes  of  identification  than  of  classification. 

How  far  is  this  conclusion  borne  out  by  a  study  of  the 
relation  between  fermentation  tests  and  agglutination  re- 
actions ? 

In  some  cases,  alteration  in  fermenting  powers  is  not 
accompanied  by  any  disturbance  in  the  agglutination  pro- 
perties,  which  remain  constant. 

For  example,  Twort  and  Penfold  have  both  shown  that  the 
altered  fermenting  power  on  the  part  of  B.  typhosus  towards 
lactose  is  not  accompanied  by  any  alteration  in  agglutination 
properties.  The  organism  described  by  Klotz  as  differing  from 
B.  typhosus  in  its  capacity  to  ferment  lactose  and  saccharose, 
and  in  other  characters,  nevertheless  was  agglutinated  by 
typhoid  serum  in  high  dilutions. 

Bahr,  in  describing  the  altered  fermenting  power  of  dy- 
sentery bacilli,  following  transmission  through  the  intestine  of 
the  fly,  states  that  the  variants  displayed  no  alteration  in 
agglutination  properties.  Lentz  mentions  a  "Flexner"  strain 
which  after  seven  years'  laboratory  cultivation  lost  its  power  to 
ferment  maltose  but  still  retained  its  agglutination  properties 
unchanged. 

In  other  cases,  alteration  in  fermenting  power  sis  associated 
with  loss  of  agglutination  properties.  For  example,  Wilson, 
in  describing  an  organism  isolated  from  the  urine  of  a  "typhoid 
carrier"  and  considered  by  him  to  be  a  derivative  of  B.  typho- 
sus,  states  that  not  only  were  the  fermenting  properties  altered 
but  the  agglutination  tests  no  longer  corresponded  with  those 
of  B.  typhosus.  Penfold  found  that  colonies  of  B.  typhosus 
which  had  lost  the  property  of  fermenting  glycerine,  showed 
impaired  agglutinability  also,  though  typical  fermenting 
colonies  on  the  same  plate  were  normal  as  regards  agglutina- 
tion. 

Horrocks  observed  that  a  strain  of  B.  typhosus  derived 
from  the  urine  of  a  carrier,  when  grown  in  the  diluted  filtered 


CH.  v]    VARIATIONS  IN  FERMENTING  POWER         69 

urine  of  a  second  "  typhoid  carrier,"  was  deprived  not  only  of 
its  power  to  ferment  but  also  its  property  of  agglutinating  in 
the  presence  of  typhoid  serum. 

Thirdly,  classifications  according  to  fermenting  powers  and 
according  to  agglutination  proper  ties  give  altogether  different 
results. 

Ohno  (1906),  in  his  elaborate  investigations  on  74  strains 
of  dysentery  derived  from  different  sources,  found  that  a 
classification  based  on  their  different  powers  of  fermentation 
did  not  correspond  with  a  classification  according  to  their 
agglutination  reactions.  Torrey  describes  dysentery  bacilli 
possessing  different  fermenting  properties  but  giving  the  same 
agglutination  reactions.  The  pneurnococcus  is  distinguished 
from  other  members  of  the  streptococcus  group  by  its  different 
fermenting  properties,  particularly  with  reference  to  inulin; 
nevertheless  the  pneumococcus  and  other  streptococci  tend  to 
originate  common  group  agglutinins. 

It  would  appear  therefore  that  the  power  of  producing 
fermentation  bears  no  relation  to  agglutinability,  and  in  the 
final  resort,  if  reliance  is  to  be  placed,  for  purposes  of  classifica- 
tion, on  one  test  the  other  must  necessarily  be  discredited. 
As  regards  identification,  however,  there  is  this  to  be  said — 
organisms  immediately  after  their  isolation  from  the  tissues 
or  excretions  are,  as  a  rule,  more  typical  in  their  fermenting 
properties  than  the  same  organisms  after  even  a  short  period 
on  artificial  media,  whereas  the  reverse  frequently  holds  true 
as  regards  their  agglutination  reactions.  In  deciding,  there- 
fore, which  of  the  two  series  of  tests  is  to  be  relied  upon  in 
cases  where  they  conflict,  the  length  of  the  period  of  cultivation 
is  of  great  importance ;  the  fermentation  tests  will  be  found 
most  reliable  in  the  identification  of  an  organism  in  those 
cases  where  agglutination  tests  are  least  so. 

It  is  seen  then  that  the  fermentation  tests,  though  for 
purposes  of  classification  they  may  prove  at  variance  with 
the  agglutination  reactions,  as  a  means  of  identification  may 
supplement  the  latter. 


70        VARIATIONS  IN  FERMENTING  POWER    [CH.  v 


THE  VALUE  OF  VARIATIONS  IN  THE  SUGAR  REACTIONS 
IN  THE  IDENTIFICATION  OF  BACTERIA. 

There  are  ways  in  which  the  actual  variations  in  the  sugar 
reactions  may  be  of  value  in  identifying  an  organism. 

In  the  first  place,  the  variations  observed  may  themselves 
be  specific  in  character  and  so  far  from  obscuring  the  identity 
of  an  organism  may  in  some  cases  actually  contribute  to  its 
recognition,  in  the  same  way  that  the  morphological  variations 
of  B.  diphtheriae  help  to  establish  its  identity. 

In  the  second  place  the  variations  may  help  to  identify 
the  source  of  the  organism  by  indicating  the  nature  of  its 
recent  environment.  Thus,  if  "passage"  is  found  to  modify 
the  fermenting  power  of  a  particular  species  of  bacteria  in 
a  particular  direction,  then  such  modification  when  found  to 
exist  in  a  member  of  that  species  may  indicate  a  recent  animal 
host.  Again,  if  growth  in  a  certain  material  is  known  to  lead  to 
certain  modified  "sugar  reactions"  on  the  part  of  a  particular 
species,  then  such  modification,  when  it  is  found,  may  furnish 
a  clue  to  the  source  of  the  organism  in  question.  For  example, 
it  has  been  pointed  out  by  several  observers  (Re vis,  1908)  that 
the  saccharose-fermenting  type  of  the  colon  bacillus  is  more 
often  isolated  from  milk  than  from  cow-dung  and  MacConkey 
has  stated  (quoted,  ibid.)  that  the  more  prolonged  the  sojourn 
in  the  former  medium  is,  the  more  prolonged  is  the  power  to 
ferment  saccharose  on  the  part  of  the  organism.  Revis  also 
describes  a  strain  of  bacteria  which  failed  to  ferment  saccharose 
when  isolated  from  cow-dung,  but  did  so  after  being  cultured 
in  milk.  Again,  strains  of  B.  coli  exhibiting  variation  in  gas 
production  are  more  commonly  of  urinary  than  of  intestinal 
origin. 

This  question  will  be  further  considered  in  a  later  chapter 
(vide  p.  144). 


CHAPTER  VI 

VARIATIONS  IN  YIKULENCE 

THE  pathologist  is  apt  to  forget  that  the  vast  majority  of 
bacteria  are  non-pathogenic,  that  is  to  say,  they  are  harmless 
to  man.  Not  only  so,  but  the  activities  of  many  of  them  are 
as  beneficial  to  him  as  those  of  the  pathogenic  bacteria  are 
the  reverse.  The  purification  of  sewage  and  the  mineraliza- 
tion of  dead  vegetable  matter,  to  mention  only  two  instances 
of  bacterial  action,  are  processes  which  contribute  to  the 
health  and  survival  of  the  human  race  no  less  than  the 
processes  of  disease  conduce  to  its  decay. 

The  power  to  cause  disease  depends  upon  two  factors.  In 
the  first  place,  it  depends  upon  the  ability  of  the  organisms 
to  become  parasitic,  that  is  to  say,  to  invade  the  living 
tissues  and  live  and  multiply  there,  and,  in  the  second  place, 
it  depends  upon  the  result  of  their  activity,  more  especially 
as  regards  the  formation  of  poisons  or  toxins. 

The  harmless  nature  of  most  bacteria  is  due  to  the  fact 
that  they  have  not  acquired  the  power  of  becoming  parasitic. 
In  some  instances,  where  bacteria  do  succeed  in  invading 
the  tissues,  the  result  of  their  activity  within  the  body  is 
apparently  harmless.  Ford  (1900)  examined  the  liver  and 
kidneys  of  healthy  animals  after  death,  with  the  most  stringent 
precautions  against  contamination,  and  found  at  least  80  per 
cent,  contained  bacteria  of  various  kinds.  In  other  cases, 
though  organisms  fail  to  invade  the  living  tissues,  they 
nevertheless  produce  symptoms  of  disease  by  manufacturing 
toxins  which  are  absorbed,  as,  for  example,  in  puerperal 
sapraemia. 

The  results  produced  by  the  presence  of  bacteria  in  the 
tissues  are  due  to  a  variety  of  causes,  which  include  (a)  the 
metabolism  of  the  living  organisms,  that  is  to  say,  the 


72  VARIATIONS  IN  VIRULENCE  [CH.  vi 

substances  assimilated  and  excreted  by  them ;  (b)  the  dis- 
integration of  the  dead  organisms  ;  (c)  the  mechanical  effect 
of  their  presence  whether  living  or  dead ;  (d)  the  response 
made  by  the  living  tissues  to  these  various  stimuli. 

The  first  factor  in  the  production  of  disease — the  "in- 
vasiveness  "  of  the  organism — is  dependent  upon  the  degree 
of  "mobility"  the  particular  organism  possesses  under  various 
conditions. 

The  second  factor — the  result  of  their  activity  within  the 
body — may  be  considered  under  two  heads  : — (1)  the  produc- 
tion of  particular  lesions  in  the  tissues  and  the  exhibition 
of  a  certain  train  of  symptoms,  both  more  or  less  peculiar  to 
the  particular  organism  giving  rise  to  them — phenomena  which 
are  discussed  under  the  term  " pathogenesis,"  and  (2)  the 
production  of  a  condition  of  "toxaemia,"  resulting  in  a 
general  impairment  of  health  and  leading  eventually  to  the 
death  of  the  body,  which  we  now  propose  to  consider  under 
the  term  "virulence." 

The  virulence  of  organisms  is  known  to  vary  under  different 
circumstances  within  wide  limits. 

1.  Thus,  cases  of  disease  which  occur  at  the  end  of  an 
epidemic  are  frequently,  though  not  invariably,  less  severe 
than  those  at  the  beginning,  the  virulence  of  the  infecting 
organism  having  gradually  diminished  during  the  course  of 
the  epidemic.  Thomson  mentions  an  epidemic  of  cerebro- 
spinal  fever  comprising  30  cases,  all  of  which  were  admitted 
to  hospital  and  received  the  same  treatment ;  of  the  first  16 
cases  admitted  all  died  except  two,  of  the  last  14  admitted  all 
lived  except  two. 

This  difference  in  virulence  might  of  course  be  apparent 
and  not  real,  the  lessened  intensity  of  the  disease  being 
accounted  for  by  a  difference  in  the  resistance  of  those 
attacked  by  it.  The  weakest  individuals,  who  are  the  first  to 
succumb  to  the  infection,  also  offer  the  poorest  defence, 
whereas  in  the  later  stages  the  less  fit  have  already  been 
weeded  out  and  the  more  robust  alone  remain  to  be  attacked, 
and  these^offer  a  more  stubborn  resistance.  Such  an  explana- 
tion might  hold  good  in  the  case  of  a  strictly  localised 


CH.  vi]  VARIATIONS  IN  VIRULENCE  73 

outbreak — for  example  on  board  a  ship  or  in  a  military 
camp — but  in  a  widespread  epidemic — in  a  crowded  city,  for 
instance, — both  weak  and  strong  individuals  are  exposed 
equally  to  infection  as  the  disease  extends. 

2.  Again,  the  same  disease  may  either  take  the  form  of 
an  epidemic  or  occur  sporadically  in  the  form  of  isolated 
cases,  apparently  unconnected  with  each  other.   In  speaking 
of  meningococcal  meningitis,  Koptik  attributes  the  diminished 
infectivity  of  the  sporadic  types  to  the  senility  and  weakened 
virulence  of  the  organism  concerned. 

3.  A  similar  diminution  in  virulence  is  observed  in  the 
case  of  some  endemic  diseases  in  the  course  of  many  genera- 
tions.   Bahr  (1912)  quotes  evidence  to  show  that  in  Fiji, 
dysentery  25  years  ago  was  a  much  more  virulent  disease 
than  it  is  at  the  present  time.   The  virulence  of  the  specific 
virus  of  syphilis  has  been  modified  considerably  in  those  parts 
of  Europe  in  which  it  has  been  prevalent  for  centuries,  such 
as  Spain. 

Here  again  the  development,  by  those  exposed  to  infection, 
of  increased  powers  of  resistance  or  "immunity,"  no  doubt 
plays  a  part,  for  when  the  infection  is  introduced  from  places 
where  it  has  long  been  prevalent  to  places  where  previously 
it  has  never  been  met  with,  the  disease  may  from  the  first 
assume  a  virulent  type. 

4.  Epidemics  of  a  particular  disease  vary  in  virulence  at 
different  times  and  at  the  same  time  but  in  different  places. 
Typhoid  fever  is,  as  a  rule,  much  less  severe  in  England  than 
the  same  disease  in  the  tropics  or  in  the  temperate  regions 
of  South  America,  although  the  organisms,  apart  from  the 
question  of  virulence,  appear  to  be  identical. 

5.  Again  a  particular  species  of  organism  may  produce, 
at  different  times,   diseased  conditions  widely  differing  in 
their  intensity.  The  classical  example  of  this  is  the  strepto- 
coccus pyogenes  which  may  produce  at  one  time  merely  a 
local  suppuration,  at  another  a  spreading  erysipelas  and  at 
another  a  rapidly  fatal  septicaemia, 

6.  Many  pathogenic  organisms  when  grown  outside  the 
body,  under  various  abnormal  conditions,  lose  their  virulence. 


74  VARIATIONS  IN  VIRULENCE  [OH.  vi 

(a)  Pasteur  showed  30  years  ago  that  B.  anthracis, 
if  grown  at  a  temperature  of  43 '5°  C.,  lost  its  virulence  in  3 
or  4  weeks.  Hewlett  and  Knight  (1897)  destroyed  the  viru- 
lence of  a  strain  of  diphtheria  bacilli  by  subjecting  it  for  17 
hours  to  a  temperature  of  45°  C.  Muir  and  Ritchie  state  that 
a  broth  culture  of  the  diphtheria  bacillus  if  exposed  for  only 
one  hour  to  a  temperature  of  65°  C.  is  rendered  much  less 
toxic  while  a  culture  of  the  tetanus  bacillus  under  the  same 
conditions  is  deprived  altogether  of  toxicity.  The  bacillus  of 
"  blackleg  "  can  likewise  be  rendered  innocuous  by  exposure 
to  a  high  temperature  (Mohler  and  Washburn,  1906). 

It  has  been  thought  that  recovery  from  infectious  diseases, 
such  as  the  exanthemata,  might  be  due  to  the  effect  produced 
in  this  way  on  the  infecting  organisms  by  the  continued  fever 
which  their  presence  provokes.  That  the  increase  in  tempera- 
ture may  be  a  protective  measure  on  the  part  of  the  body  is 
suggested  by  the  experiments  of  Lowey  and  Richter  (1897), 
in  which  the  resistance  of  rabbits  to  infection  by  the  pneumo- 
coccus,  the  diphtheria  bacillus  and  the  hog  cholera  bacillus 
was  artificially  increased  by  injury  to  the  corpus  striatum 
and  a  consequent  rise  in  temperature,  before  inoculation. 

These  observations  do  not  prove  that  the  rise  in  tempera- 
ture lessens  the  virulence  of  the  organisms.  Indeed  this 
opinion  has  been  proved  to  be  erroneous,  in  some  cases  at 
least,  by  the  work  of  Leutscher  (1911),  who  tested  the 
virulence  of  pneumococci  isolated  from  the  affected  area  of 
the  lung  at  different  stages  of  an  acute  lobar  pneumonia.  He 
found  that  the  virulence  of  the  organisms  isolated  at  the 
period  immediately  preceding  the  crisis  was  even  greater 
than  that  of  those  isolated  in  the  early  stages  of  the  disease. 

Other  observers,  working  along  different  lines,  have  shown 
that  a  comparatively  high  temperature  is  not  necessarily 
inimical  to  virulence.  Eyre,  Leatham  and  Washbourn  (1906) 
quote  the  observations  of  Kruse  and  Pansini,  which  their 
own  work  confirms,  to  the  effect  that  the  virulence  of  the 
parasitic  pneumococcus  is  often  associated  with  inability  to 
grow  at  a  temperature  much  below  that  of  the  body — 37°  C. 
A  slightly  virulent  strain  which  would  grow  readily  at  20°  C. 


CH.  vi]  VARIATIONS  IN  VIRULENCE  75 

could,  they  found,  be  converted  by  "passage"  into  a  highly 
virulent  strain  which  would  not  grow  at  a  temperature  below 
37°  C.  By  artificial  culture  the  reverse  change  could  be 
brought  about.  In  one  experiment  a  single  inoculation 
into  an  animal  sufficed  to  bring  about  the  conversion  of 
one  type  into  the  other,  the  relationship  between  virulence 
and  the  temperature  at  which  growth  would  occur  being 
constant. 

(6)  Another  abnormal  condition  of  growth  which  tends 
to  modify  the  virulence  of  organisms  is  the  presence  of  weak 
antiseptics.  Thus  Chamberland  and  Roux  (quoted,  Muir  and 
Ritchie)  found  experimentally  that  B.  anthracis  lost  virulence 
if  grown  on  a  medium  to  which  carbolic  acid  had  been  added, 
in  the  proportion  of  1  to  600,  or  a  minute  quantity  of  Pot. 
bichromate.  A  virulent  strain  of  B.  diphtheriae  is  promptly 
attenuated  by  the  addition  of  iodine  trichloride  to  the  medium 
(Mohler  and  Washburn,  1906). 

In  the  living  body  certain  secretions  play  a  similar  r61e. 
Leutscher  (1911)  proved  that  the  saliva  had  a  bactericidal 
effect  on  the  pneumococcus  and  he  attributes  the  diminished 
virulence  of  the  pneumococci  found  in  the  mouth  to  this 
agency.  Savage  showed,  by  experiment  on  himself,  that  the 
streptococcus  mastitidis,  which  causes  mastitis  in  cows,  had 
its  virulence  greatly  reduced  by  2  or  3  days'  residence  in  the 
mucous  membrane  of  the  human  pharynx. 

On  the  other  hand  the  addition  to  a  medium  of  certain 
substances  may  cause  heightened  virulence.  The  bacillus 
of  "blackleg,"  rendered  avirulent  by  exposure  to  a  high 
temperature,  has  its  virulence  completely  restored  if  lactic 
acid  is  added  to  the  medium  in  which  it  is  growing  (Mohler 
and  Washburn,  1906).  Many  organisms,  also,  which  lose  their 
virulence  rapidly  on  ordinary  culture  media,  maintain  it  for 
long  periods  when  grown  in  the  secretions  of  the  body — for 
example,  in  urine  (vide  p.  19). 

(c)  The  presence  or  absence  of  oxygen  is  another  factor 
of  importance.  For  example,  Haffkine  (quoted  Hankin,  1892) 
found  that  the  cholera  spirillum  lost  virulence  considerably 
when  grown  in  a  current  of  sterile  air,  while  Hueppe  (quoted 


76  VARIATIONS  IN  VIRULENCE  [CH.  vi 

Adami,  1892)  observed  that  its  virulence  was  heightened  by 
anaerobic  growth. 

Pasteur,  when  investigating  cultures  of  chicken  cholera, 
found  that  their  virulence  gradually  disappeared,  but  he 
discovered  that  it  was  maintained  if  he  grew  the  organisms 
in  sealed  tubes  so  that  oxygen  was  excluded.  By  this 
procedure  loss  of  moisture  was  likewise  prevented  and  this 
fact  may  possibly  have  been  of  no  less  importance  than  the 
exclusion  of  air. 

Other  organisms  which,  normally,  do  not  readily  lose  viru- 
lence, do  so  rapidly  if  grown  in  an  atmosphere  of  compressed 
air  (Muir  and  Ritchie). 

Harass  (1906)  succeeded  in  growing  certain  "anaerobic" 
bacteria  in  the  presence  of  air  and  he  found  that  under  these 
conditions  the  bacillus  of  malignant  oedema  lost  virulence 
though  the  bacillus  botulinus  retained  it.  It  is  well  known 
that  the  bacillus  of  diphtheria  produces  toxins  more  plentifully 
when  there  is  an  abundant  supply  of  air  (Clark,  1910). 

(d)  In  other  cases  the  loss  of   virulence  on   artificial 
cultivation  is  to  be  attributed  to  the  influence  of  sunlight, 
which  is  known  to  have  this  effect  on  B.  antliracis  and  other 
pathogenic  bacteria  (Marshall  Ward  and  Blackman,  1910). 

(e)  The  reaction  of  the  medium  may  also  influence  viru- 
lence.  Miss  Peckham  (1897)  found  that  the  addition  of  an 
alkali  to  the  medium  increased  the  virulence  of  a  strain  of 
B.  coli.  Undue  acidity  of  the  medium  may  result  from  the 
action  of   the  organisms  themselves    in    splitting    up    the 
carbohydrate  present. 

7.  Practically  every  organism  becomes  less  virulent  when 
cultivated  for  any  length  of  time  outside  the  body,  that  is  to 
say,  on  artificial  media,  even  under  the  most  favourable 
conditions.  The  common  pus  cocci  and  the  pneumococcus 
afford  good  illustrations  of  this.  The  loss  of  virulence  occurs 
even  when  the  growth  is  abundant  and  it  persists  on  sub- 
culture. 

Possibly  some  of  the  factors  responsible  for  this  change 
are  those  just  mentioned,  namely  differences  in  temperature, 
the  presence  of  oxygen,  exposure  to  sunlight,  the  increased 


CH.  vi]  VARIATIONS  IN  VIRULENCE  77 

acidity  of  the  medium.  Other  contributing  factors  are  found, 
no  doubt,  in  the  nature  of  the  medium  itself — both  as  regards 
its  chemical  composition  and  its  physical  properties. 

(a)  The  difference  in  chemical  composition  between  the 
body  fluids  and  laboratory  media  must  necessarily  profoundly 
influence  the  metabolism  of  organisms  transferred  from  one 
to  the  other.  The  more  closely  the  artificial  medium  used 
resembles  chemically  the  body  fluids,  the  less  influence  will 
this  factor  exert.  For  example,  on  solidified  blood  serum,  or 
media  to  which  blood  has  been  added,  or  ascitic  fluid,  viru- 
lence is  maintained  for  a  greater  length  of  time  than  on  other 
media.  Eyre  and  Washbourn  (1899)  found  that  the  parasitic 
pneumococcus  kept  its  virulence,  undiminished  for  a  couple 
of  months  on  blood  agar  but  not  on  ordinary  media.  Anne 
Williams  (1902)  found  many  strains  of  diphtheria  bacillus, 
which  were  quite  non-pathogenic  when  inoculated  from  broth, 
were  highly  toxic  when  inoculated  from  serum  culture  or 
ascitic  broth. 

In  the  case  of  pathological  exudations  the  question  of 
chemical  composition  is  of  even  greater  significance,  for  the 
composition  of  these  fluids  is  dependent  on  processes  of  great 
complexity  and  differs  widely  from  that  of  healthy  excretions, 
and  it  is  apparently  by  virtue  of  this  very  difference  that 
pathological  exudations  possess  the  power  of  maintaining 
the  virulence  of  organisms  growing  in  them.  It  is  found  in 
practice  that  the  best  fluid  in  which  to  preserve  organisms 
from  a  pathological  source  unchanged  for  subsequent  examina- 
tion is  normal  saline  to  which  a  considerable  quantity  of  the 
infected  material  itself  has  been  added,  whether  it  be  blood 
or  pus  or  fluid  from  a  serous  cavity  or  some  other  secretion. 
Horrocks  took  the  urine  of  a  "typhoid  carrier"  which  was 
loaded  with  virulent  bacilli  and  kept  it  for  12  months  in 
flasks  exposed  to  the  light  and  frequently  opened  to  the  air. 
At  the  end  of  that  time  he  found  that  the  bacilli  were  as 
virulent  as  when  first  examined,  though,  when  subcultured 
on  to  agar  or  into  broth,  virulence  was  rapidly  lost.  Thus, 
two  strains  of  virulent  typhoid  bacilli  from  the  urine  of 
"  Carrier  /"  and  "  Carrier  S"  failed  to  kill  a  guineapig,  when 


78  VARIATIONS  IN  VIRULENCE  [CH.  vi 

injected  into  the  peritoneal  cavity,  after  being  cultivated  on 
agar  for  periods  of  two  weeks  and  three  weeks  respectively. 

A  striking  example  of  the  influence  exerted  by  the  culture 
medium  is  afforded  by  the  observation  that  vaccination  with 
a  plague  strain  grown  on  agar  will  protect  rats  against  itself 
but  not  against  the  same  strain  grown  on  serum  (quoted 
Penfold,  1914). 

(b)  The  artificial  or   "unnatural"  physical  conditions 
under  which  laboratory  cultures  grow  are  no  less  important. 
The  difference  between  a  medium  of  solidified  blood  serum 
and  the  blood  circulating  in  the  living  body  is  not  only  one 
of  chemical  composition.   When  grown  on  solid  media  the 
organisms  are  crowded  together  and  there  must  necessarily 
be  an  accumulation  and  concentration  of  acids  formed  from 
the  medium,  and  also  of  excreted  toxins  in  their  immediate 
vicinity,  which  may  inhibit  their  power  to  produce  more  of 
the  latter  substances. 

If  in  this  case  the  metabolism  of  the  organisms  could  be 
"  damped  down,"  the  possibly  inhibitory  influence  of  such  an 
accumulation  might  be  prevented.  Shiga  (quoted  by  Bahr, 
191 2)  found  that  the  virulence  of  his  dysentery  bacillus  could 
be  maintained  for  as  long  as  12  months  by  keeping  the 
cultures  at  freezing  temperature.  Whether  decreased  meta- 
bolism is  the  true  explanation  in  this  case  or  not,  it  is 
impossible  to  say  without  further  investigation. 

In  the  living  body,  on  the  other  hand,  the  products  of  the 
metabolism  of  the  organism  are  dissolved  and  carried  away 
and  absorbed  and  excreted. 

(c)  Moreover,  in  the  living  tissues  the  presence  of  toxins 
provokes  the  formation  or  liberation  of  "  immune  bodies  "  as 
a  protective  measure  on  the  part  of  the  body,  and  it  has  been 
shown  by  Ainley  Walker  (1903)  that  the  influence  of  these 
"  immune  bodies  " — whatever  their  exact  nature  may  be — is 
to  heighten  the  virulence  of  the  organism  which  led  to  their 
production.   This  observer  states,  with  regard  to  B.  typhosus, 
that  "  the  result  of  growing  the  bacillus  in  its  immune  serum 
was  a  diminution  in  its  agglutinability,  a  heightening  of  its 
virulence  and  an  increase  in  its  resistance  to  serum  protection." 


CH.  vi]  VARIATIONS  IN  VIRULENCE  79 

Substances  which  increase  the  virulence  of  an  organism 
may  be  formed  in  the  tissues  as  a  result  of  infection  by  an 
organism  of  a  different  species.  For  example,  the  staphylo- 
coccus  aureus  produces  more  extensive  local  lesions  if,  with 
the  organism,  is  injected  a  small  quantity  of  the  serum  from 
a  case  of  spreading  cellulitis;  if  the  local  exudate  from  a 
cellulitis  is  substituted  for  the  serum  no  such  effect  is  produced, 
showing  that  the  phenomenon  is  due  to  bodies  formed  not  by 
the  bacteria  which  cause  the  cellulitis  but  by  the  tissues. 
Moreover  the  serum  from  a  case  of  spreading  cellulitis  has 
the  same  effect  in  the  case  of  other  kinds  of  infection — due 
to  the  pneumococcus,  B.  typhosus,  the  tubercle  bacillus  and 
cholera  (Hektoen). 

(d)  Yet  a  fourth  contributing  factor  to  the  loss  of  virulence 
on  artificial  media  is  the  fact  that  other  organisms  are,  as  far 
as  possible,  excluded.  The  object  of  the  investigator  is  to 
obtain  a  pure  culture,  by  the  use  of  selective  media  or  by 
subculturing.  In  pathological  secretions  the  primary  infecting 
organism  grows  in  the  presence  of  many  other  saprophytic 
or  parasitic  bacteria.  A  typhoid  stool  contains  a  multitude 
of  organisms  in  addition  to  Eberth's  bacillus  and  pneumonic 
sputum  often  supplies  evidence  of  a  mixed  infection.  The 
influence  of  symbiosis  on  the  virulence  of  organisms  will  be 
referred  to  later,  but  it  is  noteworthy,  in  this  connection,  that 
many  organisms  which  are  more  prone  than  others  to  lose 
virulence  when  grown  on  artificial  media  are  likewise  more 
often  found  in  pathological  conditions  associated  with  other 
organisms. 

Whatever  the  explanation  may  be,  the  fact  remains  that 
cultures  on  artificial  media  tend  to  lose  virulence.  On  the 
other  hand  if  bacteria  grow  in  pathological  secretions,  and 
particularly  if  they  successfully  invade  the  body  of  an  animal 
and  multiply  in  that  animal's  tissues,  their  virulence  often 
becomes  greatly  increased. 

8.  Pathological  secretions  possess  the  power  not  only  of 
preserving  virulence  but  of  actually  developing  that  property 
on  the  part  of  organisms  growing  in  them.  For  example,  the 
comparatively  harmless  B.  coli  communis,  present  in  large 


80  VARIATIONS  IN  VIRULENCE  [CH.  vi 

numbers  in  the  healthy  intestine,  develops  in  many  inflam- 
matory conditions  of  the  intestinal  mucous  membrane  the 
property  of  virulence.  Sanarelli  (1894)  caused  the  colon 
bacillus  to  become  virulent  in  experimental  typhoid  fever 
by  producing  an  inflammation  with  typhoid  toxin.  Acute 
peritonitis  due  to  B.  coli,  following  strangulation  of  the  gut — 
a  sequence  frequently  observed  clinically — would  appear  to 
depend  upon  something  more  than  a  lowering  of  the  vitality 
of  the  tissues,  for  De  Klecki  (quoted  by  Peckham,  1897)  found, 
by  experimenting  on  dogs,  that  the  colon  bacillus  acquired 
virulence  in  the  lumen  of  a  strangulated  coil  of  intestine. 
Dreyfuss  has  described  the  increased  virulence  of  B.  coli  in 
intestinal  disease,  and  Fermi  and  Salto  (quoted,  Peckham) 
a  similar  increased  virulence  in  inflamed  conditions  of  the 
intestines  due  to  cold,  bad  food,  etc. 

Harris  (1901)  tested  the  toxicity  of  29  strains  of  B.  coli 
communis  derived  from  various  sources.  Out  of  15  strains 
obtained  from  "natural  sources'"'  (healthy  faeces,  sewage, 
water,  milk,  shellfish)  only  two  were  virulent  (one  of  these  very 
slightly  so)  whereas  out  of  11  strains  derived  from  pathological 
secretions  (pus  and  the  stools  in  epidemic  cholera,  cholera 
nostras  and  summer  diarrhoea)  only  one  (from  the  last-named 
source)  was  non-virulent. 

The  acquirement  of  virulence  by  saprophytic  organisms  in 
the  cavity  of  an  inflamed  uterus  during  the  puerperium,  is 
another  case  in  point. 

9.  In  many  cases  the  development  of  virulence  by  organisms 
growing  in  pathological  exudations  is  due  not  only  to  the 
presence  of  inflammatory  products  or  to  the  absence  of  sub- 
stances normally  found  in  the  healthy  secretions  but  also  to 
some  extent,  as  we  have  already  said,  to  the  presence  of  other 
bacteria  and  their  products. 

In  discussing  the  effect  of  symbiosis  on  organisms,  reference 
was  made  (vide  p.  22)  to  the  fact  that  substances  excreted  by 
one  species  of  bacteria  may  markedly  influence  the  growth  of 
another  species.  An  example  was  there  given  of  a  strain  of 
B.  influenzae  which  would  grow  on  a  sterilised  medium 
previously  impregnated  with  the  products  of  a  staphylococcal 


CH.  vi]  VARIATIONS  IN  VIRULENCE  81 

growth   but  which  could  not  be  made  to  grow  otherwise 
(Allen,  1910). 

That  symbiosis  is  an  important  factor  in  determining  not 
only  the  growth  but  also  the  virulence  of  a  strain  of  bacteria, 
is  abundantly  proved  both  by  experiment  and  observation. 

It  is  said  that  a  dog  will  not  succumb  to  the  infection  of 
tetanus  unless  it  is  infected  simultaneously  with  pyogenic  cocci 
and  in  man  it  is  recognised  that  the  prognosis  is  more  serious 
if  the  tetanus  gains  access  to  the  body  by  means  of  a  sup- 
purating wound.  Some  authorities  explain  these  facts  by 
assuming  that  the  tetanus  bacillus  can  only  multiply  in  the 
body  in  the  presence  of  pus-forming  cocci  (Marshall  Ward 
and  Blackman,  1910),  but  analogy  with  other  phenomena  of 
the  same  kind  certainly  suggests  that  it  is  a  question  of  altered 
virulence. 

Sanarelli  observes  that  B.  coll  communis  in  typhoid  stools 
was  highly  virulent.  Muir  and  Ritchie  state  "guinea-pigs 
may  resist  the  subcutaneous  injection  of  a  certain  dose  of  the 
typhoid  bacillus,  but  if  at  the  same  time  a  sterilised  culture  of 
the  bacillus  coli  be  injected  into  the  peritoneum  they  quickly 
die  of  general  infection."  These  authors  attribute  the  pheno- 
menon to  the  diminished  vitality  of  the  animal,  but  here 
again  analogy  suggests  that  increased  viability  or  heightened 
virulence  on  the  part  of  the  typhoid  bacilli  may  be  factors  of 
no  less  importance. 

Other  examples  of  symbiosis  influencing  virulence  will  be 
given  in  discussing  "passage." 

10.  The  successful  invasion  of  the  animal  body  by  bacteria, 
leading  to  their  increased  virulence,  may  be  brought  about 
experimentally. 

Pasteur  was  the  first  to  discover  that  virulence  could  be 
"exalted"  by  "  artificial  passage"  through  an  animal  or  series 
of  animals.  Rabbits  or  guinea-pigs  are  those  commonly  em- 
ployed for  the  purpose  and  inoculation  may  be  made  into  the 
blood  stream  or  the  peritoneal  cavity  and  a  cultivation  made 
subsequently  from  the  heart's  blood  or  the  peritoneal  fluid ; 
or  the. organisms  may — instead  of  being  introduced  directly 
into  the  peritoneum — be  shut  up  in  a  closed  sac  which  is  then 
D.  6 


82  VARIATIONS  IN  VIRULENCE  [OH.  vi 

inserted  into  the  body  cavity  and  allowed  to  remain  there  for 
a  certain  time. 

By  the  last  named  method,  for  example,  Martin  (1898) 
increased  the  virulence  of  a  slightly  virulent  strain  of  B. 
diphtheriae. 

Eyre  and  Washbourn  (1899)  showed  that  the  saprophytic 
pneumococcus,  found  in  the  mouths  of  healthy  persons,  could 
by  "passage"  be  made  as  virulent  as  the  parasitic  type  isolated 
from  an  acute  lobar  pneumonia.  The  number  of  inoculations 
required  varied  considerably  in  different  cases.  In  most  of  the 
experiments  a  series  of  eight  or  ten  rabbits  sufficed.  In  one 
case  virulence  only  reached  its  height  after  no  less  than 
53  passages.  In  another  case  a  single  inoculation  was  sufficient 
to  convert  an  avirulent  organism  into  a  highly  virulent 
one.  They  noted  that  strains  in  which  virulence  was  easily 
raised  were  able  to  maintain  their  exalted  virulence  on 
suitable  media  for  a  long  time,  while  those  strains  which 
very  slowly  acquired  virulence,  quickly  lost  it  on  artificial 
culture. 

Mohler  and  Washburn  (1906)  mention  that  the  virulence 
of  the  virus  of  rabies  is  increased  by  passage  through  rabbits 
and  that  the  cholera  organism,  after  passage  through  guinea- 
pigs,  becomes  much  more  virulent  towards  pigeons. 

Other  animals  than  those  named  may  be  utilised  for  the 
purpose  of  "passage."  For  example,  Salter  (1899),  by  means 
of  five  successive  passages  through  the  goldfinch,  raised  the 
virulence  of  the  "pseudo-diphtheria  bacillus"  sufficiently  to 
render  it  fatal  to  guineapigs. 

The  process  of  "passage"  may  be  even  more  effective  if 
it  be  made  to  alternate  with  culture  on  ordinary  media. 
Marmorek  (quoted,  Muir  and  Ritchie)  showed  that  the  viru- 
lence of  the  streptococcus  was  enormously  increased  by  grow- 
ing it  alternately  in  a  mixture  of  human  blood  serum  and 
bouillon  and  in  the  body  of  a  rabbit. 

11.  Inoculation  with  a  living  or  dead  culture  of  some 
other  organism  in  many  cases  intensifies  the  result.  Thus, 
Klein  (1903-4)  states  that  the  virulence  of  the  diphtheria 
bacillus  is  greatly  increased  by  inoculation  into  an  animal  if 


CH.  vi]  VARIATIONS  IN  VIRULENCE  83 

the  streptococcus  pyogenes  is  inoculated  with  it,  but  not  to 
the  same  degree  if  the  diphtheria  bacillus  is  inoculated 
alone. 

In  this  connection  Miss  Williams'  experiments  (1902)  are 
of  interest.  She  grew  two  strains  of  avirulent  (but  morpho- 
logically typical)  diphtheria  bacilli  with  a  strain  of  virulent 
streptococci  in  broth,  transplanting  every  three  or  four  days 
for  90  successive  generations,  without  producing  any  change 
in  the  virulence  of  either  organism. 

The  virulence  of  the  bacillus  of  malignant  oedema  is 
markedly  increased  if  this  organism  is  inoculated  together 
with  B.  prodigiosus,  and  that  of  the  streptococcus  if  it  is 
inoculated  with  B.  coli  communis  (Muir  and  Ritchie). 

Klein  (1903-4)  also  found  that  the  injection  of  many 
organisms  subcutaneously  (B.  coli,  B.  Gaertner,  B.  enteritidis 
sporogenes,  and  others)  enhanced  the  virulence  of  organisms 
such  as  B.  typhoms  or  F.  cholerae  growing  simultaneously  in 
the  peritoneal  cavity. 

The  exalted  virulence  thus  produced  applies  to  the  par- 
ticular species  of  animal  employed  for  passage  and  may,  as 
we  have  shown,  apply  to  another  species  also — but  this  is  not 
necessarily  the  case.  Indeed,  the  virulence  towards  other 
species  may  be  markedly  diminished.  For  example,  the  virus 
of  rabies  becomes  attenuated  by  passage  through  monkeys 
(Mohler  and  Washburn,  1906).  The  bacillus  of  swine  erysipelas, 
isolated  by  Loeffler,  after  passage  through  the  rabbit  shows 
exalted  virulence  towards  this  animal  but  attenuated  virulence 
towards  pigs  (Adami,  1892).  Duguid  and  Burdon-Sanderson 
found  that  the  virulence  of  anthrax  bacilli  for  bovine  animals 
was  diminished  after  passage  through  a  number  of  guineapigs. 
Pasteur  found  that,  if  swine  plague  were  inoculated  from  rabbit 
to  rabbit,  the  organism  became  more  virulent  for  the  rabbit 
but  less  virulent  for  pigs  (Muir  and  Ritchie).  Streptococci,  on 
being  inoculated  through  a  series  of  mice,  acquire  increased 
virulence  for  these  animals  but  become  less  virulent  for  rabbits 
(Knorr,  ibid.). 

Many  other  examples  might  be  given.  A  familiar  one  is 
the  preparation  of  the  calf  lymph  for  "vaccination"  where 

6—2 


84  VARIATIONS  IN  VIRULENCE  [OH.  vi 

advantage  is  taken  of  the  same  phenomenon  to  attenuate  the 
virus  of  smallpox. 


THE  SIGNIFICANCE  OF  VARIATION  IN  VIRULENCE. 

There  is  a  mass  of  evidence,  therefore,  to  show  that  the 
virulence  of  bacteria  is  very  variable.  What  is  the  explanation 
and  significance  of  these  observations? 

Andrewes,  in  the  Horace  Dobell  Lecture  already  quoted 
(vide  p.  3),  described  the  evolution  of  bacteria  from  harmless 
mineral  feeders  into  animal  saprophytes  and  finally  into 
parasitic  organisms  and  showed  that  virulence  was  probably 
the  latest  property  to  be  acquired  in  the  process.  It  is  an  axiom 
in  the  study  of  evolution  that  the  latest  characteristic  or 
function  to  be  acquired  is  the  most  unstable  and  the  first  to 
be  lost  if  retrogression  occurs.  Each  new  function  acquired 
indicates  a  higher  degree  of  specialisation.  The  more  highly 
specialised  the  activity  of  an  organism  becomes  the  more 
intricate  are  the  processes  upon  which  it  depends  and  the 
more  readily  is  it — like  a  complicated  mechanism — "put  out 
of  gear."  It  is  recognised,  for  example,  by  alienists  that  in  cases 
of  mental  degeneration  the  highest  faculties,  which,  from  their 
absence  or  rudimentary  character  in  lower  animals  and  on 
other  grounds,  are  regarded  as  having  been  the  last  to  be 
evolved,  are  the  first,  and  often  the  only  ones,  to  show 
signs  of  impairment.  The  loss  of  virulence  by  bacteria,  often 
unaccompanied  by  any  other  alteration  in  the  character  of 
the  organism  is  another  illustration  of  the  same  phenomenon. 

The  process  by  which  this  property  of  virulence  is  regained 
by  a  strain  of  bacteria  is  exactly  similar  to  the  process  by 
which  it  was  originally  acquired  by  the  race,  but  accelerated, 
that  is  to  say  it  is  a  case  of  the  survival  of  the  fittest. 

The  fate  however  of  a  band  of  sojdiers  raiding  an  enemy's 
country  depends  not  only  on  their  numbers  and  strength  but 
also  on  their  resourcefulness,  so  that  the  "fittest"  in  this 
connection  must  be  interpreted  to  mean  not  necessarily  the 
strongest  or  most  robust  but  those  best  able  to  protect  them- 
selves ;  and  since  the  most  successful  method  of  resistance 


CH.  vi]  VARIATIONS  IN  VIRULENCE  85 

is  to  attack,  certain  of  these  minute  organisms  acquire  the 
power  of  manufacturing  toxins  which  weaken  the  defence  and 
counteract  the  opposition  of  the  living  tissues  and  so  enable 
them  to  gain  a  firmer  foothold  there.  It  is  the  individuals 
which  thus  accommodate  themselves  to  the  exigencies  of  their 
surroundings  that  are  perpetuated  by  a  process  of  natural 
selection. 

That  it  is  a  case  of  particular  adaptation  to  environment 
and  not  merely  a  question  of  vitality  or  robustness  is  shown  by 
the  following  observations. 

(a)  "Passage"  through  a  certain  species  of  animal  while 
increasing  the  virulence  for  that  species  may  actually  diminish 
it  for  another  species.  If  the  process  merely  selected  the 
strongest,  the  strain  of  organisms  resulting  should  show 
heightened  virulence  for  other  animals  also. 

(6)  The  most  virulent  organisms  are  not  necessarily  the 
most  robust.  Eyre  and  Washbourn  (1899)  found,  as  Kruse  and 
Pansini  had  done  previously,  that  in  the  case  of  the  pneumo- 
coccus  the  exact  contrary  was  true.  "The  most  virulent  strains 
were  those  which  were  most  delicate  and  sensitive  in  artificial 
cultivations  and  the  less  virulent  ones  were  much  less  delicate 
and  could  grow  under  conditions  in  which  the  virulent  ones 
were  unable  to  flourish."  The  parasitic  type  required  a  certain 
reaction  and  temperature  and  special  media.  It  would  not 
grow  if  the  reaction  was  even  faintly  acid  or  at  a  temperature 
much  below  that  of  the  body  and  rapidly  died  out  on  agar  or 
in  broth.  The  saprophytic  type  grew  luxuriantly  either  at 
37°  C.  or  at  20°  C.,  in  broth,  agar,  potato  or  gelatin,  whether 
acid  or  alkaline,  and  retained  its  vitality  for  many  months. 

(c)  Analogy  with  other  processes  of  adaptation  lends 
further  support  to  the  view  put  forward.  Thus,  Rettger  and 
Sherrick  (1911)  have  shown  that  by  artificial  selection  a  strain 
of  organisms  can  be  made  unusually  resistant  to  the  action  of 
an  antiseptic  such  as  corrosive  sublimate.  Penfold  (1911  c) 
showed  that  natural  selection  might  in  the  same  way  develop 
a  special  power  of  resistance  to  an  antiseptic — in  this  case 
chlor-acetic  acid.  The  last-named  observer  went  further  and 
demonstrated  that  the  particular  strain  of  organisms  which 


86  VARIATIONS  IN  VIRULENCE  [CH.  vi 

showed  increased  powers  of  resistance  to  chlor-acetic  acid 
failed  to  show  any  similar  increase  in  their  power  of  resisting 
other  antiseptics  such  as  carbolic  acid  or  formaldehyde.  In 
other  words  it  was  a  case  of  adaptation  and  not  merely 
increased  robustness. 

(d)  The  acquirement  of  virulence  during  the  process  of 
passage  is  sometimes  accompanied  by  other  changes  in  the 
character  of  the  organisms  which  are  only  to  be  accounted  for 
by  the  theory  of  adaptation  to  a  particular  environment.  For 
example,  the  parasitic  pneumococcus  isolated  from  the  human 
lung  in  an  acute  lobar  pneumonia  (Eyre  and  Washbourn)  was 
characterised  by  inability  to  grow  except  at  a  temperature 
near  that  of  the  human  body.  Again  the  avian  tubercle 
bacillus — the  particular  race  of  tubercle  virulent  to  birds — 
grows  best  at  their  body  temperature  which  is  higher  than 
that  of  man,  namely  43*5°  C.,  a  temperature  at  which  human 
tubercle  dies. 

Certain  difficulties  are  nevertheless  presented  by  this 
theory  of  the  development  of  virulence  by  natural  selection. 

(a)  The  first  to  suggest  itself  is  the  fact  that  toxins  are 
"intracellular"  as  well  as  "extracellular"  and,  inasmuch  as  the 
intracellular  toxins  are  not  liberated  until  the  death  of  the 
organism  and  its  disintegration,  their  nature  and  potency  can 
obviously  have  no  influence  on  that  organism's  survival  or 
perpetuation.  We  are  not  concerned,  however,  with  one 
isolated  bacterium's  struggle  for  existence  so  much  as  with 
the  fate  of  a  host  of  bacteria  invading  the  tissues,  and  it  is  no 
less  obvious  in  their  case  that  if  their  intracellular  toxins  are 
destructive  of  the  vitality  of  the  tissues  the  living  bacteria 
will  receive  assistance  from  their  dead  and  disintegrated 
comrades  which  they  would  not  otherwise  do,  and  this  fact  may 
determine  the  success  of  their  invasion  and  consequently  their 
perpetuation. 

It  is  possible  that  in  the  case  of  some  bacteria  the  toxic 
action  of  the  substances  set  free  on  their  disintegration  is 
a  purely  physiological  one — comparable  to  the  effects  produced 
by  the  absorption  of  extravasated  blood — and  not  due  to 
a  special  adaptation.  The  protective  action,  however,  of 


CH.  vi]  VARIATIONS  IN  VIRULENCE  87 

tuberculin  and  modern  vaccines  against  specific  diseases 
suggests  that  the  role  of  the  intracellular  toxins  is  of  greater 
significance  than  this  hypothesis  would  admit. 

(6)  Another  question  that  suggests  itself  is  this:  why, 
if  successive  passages  increase  virulence,  do  not  infectious 
diseases,  which  are  continually  undergoing  this  process  of 
passage,  become  of  deadly  virulence? 

One  explanation  is  that  an  obligatory  parasite  which  kills 
its  host  sinks  the  ship  it  is  sailing  in  and  thereby  sacrifices  its 
own  chance  of  survival,  so  that  the  most  virulent  organisms 
are  weeded  out  and  destroyed.  Many  infectious  diseases, 
however,  have  not  reached  such  a  high  degree  of  virulence 
that  this  factor  can  have  become  operative  in  their  case. 

(c)  Yet  a  third  difficulty  in  accounting  for  the  development 
of  virulence  by  natural  selection  is  the  occurrence  of  pheno- 
mena such  as  that  described  by  Eyre  and  Washbourn  in  which 
an  avirulent  pneumococcus  acquired  a  high  degree  of  virulence 
after  a  single  passage  through  an  animal.  The  virulence  so 
acquired  was  maintained  for  a  couple  of  months  on  artificial 
media,  much  longer,  that  is  to  say,  than  it  was  found  possible 
to  maintain  the  same  character  when  developed  more  slowly. 
It  is  difficult  to  conceive  how,  in  the  course  of  so  few 
generations  comparatively,  natural  selection  could  cause  the 
character  of  virulence  to  predominate  to  such  an  extent. 
Moreover,  if  it  did  so  one  would  expect  the  character  to  be 
lost  with  equal  readiness  outside  the  body. 

The  explanation  may  be  that  when  avirulent  and  virulent 
pneumococci  grow  side  by  side  on  artificial  media  there  is  no 
selective  action  and  the  avirulent,  being  the  more  robust  as 
these  writers  have  shown,  soon  greatly  outnumber  the  virulent ; 
the  latter,  after  subculture  has  been  carried  out  several  times, 
may  be  so  few  in  number  that  they  give  no  evidence  of  their 
presence.  "Passage"  in  this  case,  by  eliminating  the  avirulent, 
would  very  soon  select  out  a  pure  strain  of  virulent  organisms. 

Another  explanation,  however,  suggests  itself.  The  change 
has  more  the  aspect  of  an  alteration  in  metabolism  occurring 
as  a  direct  response  to  the  change  in  the  food  material  pro- 
vided. It  is  easy  to  imagine  that,  once  established,  such 


88  VARIATIONS  IN  VIRULENCE  [CH.  vi 

altered  metabolism  might  persist  outside  the  body  for  some 
time  if  the  substances  provided  for  assimilation  remained  the 
same.  In  the  case  quoted,  Eyre  and  Washbourn  found  that 
virulence  was  maintained  only  on  media  containing  blood 
(blood  agar)  just  as  Anne  Williams  found  that  the  virulence  of 
diphtheria  bacilli  was  maintained  only  on  serum  or  ascitic 
fluid.  (The  possible  relationship  between  toxicity  and 
altered  metabolism  will  be  discussed  later.) 

(d)  Another  difficulty  is  the  development  of  the  property 
of  virulence  by  organisms  outside  the  living  tissues,  as,  for 
example,  by  saprophytic  bacteria  in  the  intestine  or  in  the 
cavity  of  the  uterus  during  the  puerperium.  What  part  can 
adaptation  or  selection  play  in  the  case  of  these  ? 

It  is  true  that  a  saprophyte  which  has  acquired  the  power 
of  excreting  toxins  has  thereby  acquired  the  power  also  of 
lowering  the  vitality  of  the  living  cells  exposed  to  their 
action,  or  even  of  killing  these  in  cases  where  the  superficial 
tissues  have  been  injured  previously.  The  toxic  saprophyte 
by  such  means  is  enabled  to  procure  fresh  food-stuffs  for  its 
own  use,  but  since  it  is  forced  to  share  the  spoils  with  its 
non-toxic  brethren  this  accomplishment  is  less  a  private  gain 
than  a  public  advantage,  and  hardly  conduces  more  to  its 
own  survival  than  it  does  to  theirs.  It  is  evident,  neverthe- 
less, that  a  strain  of  saprophytes  which  developed  toxic  pro- 
perties might  survive  and  multiply  under  conditions  in  which 
a  strain  of  non-toxic  saprophytes  would  die  out,  so  that  the 
strain  of  saprophytes  possessing  the  greatest  toxicity  would, 
other  things  being  equal,  stand  the  best  chance  of  being  per- 
petuated. 

A  saprophyte,  e.g.  in  the  uterus  during  the  puerperium, 
may  not  only  develop  extreme  toxicity  but  may  actually 
invade  the  living  tissues  and  become  parasitic.  Here  ob- 
viously other  questions  are  involved.  One  of  these  concerns 
the  part  played  by  the  food-stuffs  of  bacteria  and  the  effects 
of  changes  in  these. 

The  fortunes  of  an  invading  army  depend  as  much  upon 
its  successful  victualling  as  upon  its  armament ;  if  the  former 
breaks  down  the  latter  is  of  no  avail.  A  non-toxic  saprophyte 


CH.  vi]  VARIATIONS  IN  VIRULENCE  89 

which  develops  into  a  virulent  parasite,  invading  the  living 
tissues,  undergoes  a  twofold  adaptation,  for  it  must  neces- 
sarily acquire  the  faculty  of  nourishing  itself  upon  unac- 
customed food-stuffs  as  well  as  the  faculty  of  excreting  toxins. 
The  second  would  be  useless  without  the  first. 

What  is  the  relation  between  these  two  faculties?  Both 
imply  altered  metabolism ;  one  involves  a  change  in  assimi- 
lation, the  other  a  change  in  excretion  ;  one  necessitates  the 
assimilation  of  highly  organised  materials,  in  the  shape  of 
proteid  or  extractives,  the  other  consists  in  the  excretion  of 
complex  substances  which  in  some  cases  have  been  proved  to 
be  proteid  in  nature — the  toxalbumins — and  in  others  are 
more  akin  to  extractives. 

The  possibility  naturally  suggests  itself  that  the  second 
phenomenon  may  be  dependent  upon  the  first,  that,  in  some 
cases  at  least,  the  excretion  of  toxins  is  the  direct  result  of  the 
altered  assimilation,  comparable  to  the  increased  toxicity  of 
the  urine  of  a  man  on  a  certain  diet. 

This  hypothesis  would  go  far  towards  explaining  the 
development  of  toxicity  by  saprophytic  organisms  growing 
in  material  of  a  highly  albuminous  nature  and  rich  in  extrac- 
tives, in  an  inflamed  uterus  or  in  pathological  exudations 
wherever  found,  without  any  actual  invasion  of  the  living 
tissues  by  the  organism  taking  place. 

Two  observations  are  of  interest  in  this  connection. 

(a)  Miss  Peckham  (1897),  in  speaking  of  coliform  organ- 
isms, expresses  the  opinion  that  the  carbohydrate  con- 
stituents of  the  culture  medium  are  always  attacked  by  the 
organisms  present  in  preference  to  the  proteid  material  and 
it  is  only  when  the  supply  of  carbohydrate  is  exhausted  that 
the  proteid  is  made  use  of.  She  showed  that  if  B.  typhosus 
were  repeatedly  subcultured  in  peptone  solution  which  con- 
tained  no  carbohydrate  it  acquired  the  power  to  split  up  the 
proteid  and  produced  indol.  She  also  quotes  Pe"r4  to  the 
effect  that  the  appearance  of  the  indol  reaction  (which 
depends  upon  the  breaking  up  of  the  proteid  by  the  organisms) 
is  proof  of  the  absence  of  carbohydrate,  an  opinion  she  herself 
confirmed  by  experiment.  She  found  that  indol  formation 


90  VARIATIONS  IN  VIRULENCE  [CH.  vi 

which  followed  the  elimination  of  the  sugar  (lactose)  by  the 
bacteria  was  inhibited  by  its  further  addition. 

Glenn  (1911)  sought  to  ascertain  by  experiment  whether 
this  inhibition  in  indol  formation  was  due  to  the  acid  pro- 
duced by  the  splitting  up  of  the  sugar.  He  found,  however, 
that  even  when  the  acid  was  neutralised  indol  was  not  formed 
until  all  the  sugar  had  been  eliminated. 

It  is  interesting  to  note  that,  as  long  ago  as  1889,  Cart- 
wright  Wood  explained  the  fact  that  indol  formation  did  not 
take  place  in  the  presence  of  glycerine,  on  the  ground — not 
that  the  glycerine  interfered  with  the  activity  of  the  bacteria 
— but  that  the  glycerine  offered  them  a  pabulum  which  they 
preferred. 

(b)  The  second  observation  is  that  in  the  case  of  some 
organisms — for  example  B.  diphiheriae  (Theobald  Smith 
1899,  Fisher  1909) — toxins  are  formed  in  a  culture  only  if  the 
amount  of  the  sugar  in  the  medium  is  very  small — not  more 
than  a  "  trace." 

In  one  case,  therefore,  we  find  that  organisms  do  not  split 
up  proteid  material  as  long  as  they  can  subsist  on  other  food- 
stuffs such  as  carbohydrates,  and  in  the  second  case  we  find 
that  some  organisms,  at  any  rate,  do  not  elaborate  toxins  in 
the  presence  of  much  carbohydrate  material.  Both  these 
observations  lend  support  to  the  theory  that  the  development 
of  toxicity  may  result  from  an  alteration  in  metabolism  brought 
about  by  a  change  in  the  kind  of  food-stuffs  available. 

If  once  this  altered  metabolism  is  established  the  step  is 
a  short  one  from  a  saprophytic  existence  to  a  parasitic  one. 
The  organism  now  trained  to  feed  on  "vital"  material  has 
only  to  cross  the  border  line  between  the  dead  and  living 
tissues  to  become  a  virulent  parasite. 

The  invasion  of  the  living  tissues,  however,  on  the  part  of 
an  organism,  although  it  may  necessitate  the  altered  assimi- 
lation to  which  we  have  been  attributing  toxicity,  does  not 
always  confer  on  the  organism  the  property  of  virulence.  For 
example,  in  a  case  recorded  by  Pansini,  staphylococci  were 
repeatedly  subcultured  from  the  blood  over  a  period  of  years 
without  there  being  any  evidence  of  toxaemia. 


CH.  vi]  VARIATIONS  IN  VIRULENCE  91 

We  are  forced,  therefore,  to  the  conclusion  that  if  in  some 
cases  the  formation  of  substances  which  are  toxic  in  their 
action  is  purely  a  physiological  process  of  excretion  following 
on  altered  assimilation,  in  other  cases  this  result  is  due  to  a 
special  adaptation,  the  toxin  being  a  secretion  rather  than  an 
excretion. 

The  explanation  of  this  and  other  kindred  phenomena  is, 
however,  unsatisfactory  and  the  suggestion  has  been  made  in 
many  quarters  that  the  property  of  virulence  may  be  due 
to  the  action  of  something  more  or  less  distinct  from  the 
organism  itself  but  grafted  on  or  attached  temporarily  to  it 
— something  in  the  nature  of  a  ferment  or  enzyme.  This 
theory  we  shall  discuss  later  (vide  chap,  xi)  but  in  connection 
with  it  one  observation  will  be  made  at  this  point,  namely, 
that  if  the  liberation  or  acquisition  of  a  ferment  by  the 
organism  is  of  advantage  to  it  in  its  life  struggle,  it  may  still 
be  regarded  as  an  adaptation  to  environment  and  natural 
selection  will  in  time  cause  the  characteristic  to  predominate. 

THE  VALUE  OF  VIRULENCE  IN  CLASSIFICATION. 

In  concluding  this  section  it  only  remains  to  say  one  word 
as  to  the  value  of  classification  according  to  virulence. 
Differences  in  a  character  so  variable  as  we  have  shown 
virulence  to  be  cannot,  alone,  be  regarded  as  sufficient  to 
justify  a  separation  of  bacteria  into  distinct  "species."  The 
inconsistency  to  which  such  a  classification  gives  rise  can 
best  be  demonstrated  by  examples. 

For  instance,  we  find  in  the  throats  of  some  patients  con- 
valescent from  diphtheria,  the  so-called  "carriers,"  bacilli 
indistinguishable  from  the  Klebs-Loeffler  bacillus,  but  in 
many  cases  non-virulent.  In  such  cases  the  organism  is 
almost  certainly  the  lineal  descendant  of  the  original 
virulent  infecting  organism  and  cannot  be  regarded  as  a 
distinct  species. 

In  other  cases,  in  which  no  history  of  diphtheria  can  be 
elicited,  bacilli  again  are  found  in  the  throat  morphologi- 
cally and  culturally  indistinguishable  from  the  Klebs-Loeffler 


92  VARIATIONS  IN  VIRULENCE  [CH.  vi 

bacillus  but  non-pathogenic.  The  weight  of  evidence  is  against 
the  possibility  of  rendering  such  strains  virulent  by  passage. 
A  filtered  broth  culture,  however,  of  these  organisms  will 
provoke  the  formation  of  diphtheria  antitoxin  in  the  horse 
(Ark wright,  1909)  so  that  they  must  be  regarded  as  true 
diphtheria  bacilli  although  non-pathogenic. 

The  pneumococcus  is  found  as  a  virulent  organism  in  acute 
lobar  pneumonia.  It  is  normally  present  in  the  mouths  of 
healthy  persons  in  a  form  which  is  non-virulent  but  which 
can  be  made  virulent  by  passage  through  an  animal  and  in 
this  case  is  indistinguishable  from  the  pathogenic  variety. 
These  two  forms  are  regarded  only  as  varieties  of  the  same 
species. 

The  comparatively  harmless  B.  coli  communis,  normally 
present  in  the  human  intestine  in  health,  gives  place  in  many 
unhealthy  and  inflamed  conditions  of  the  intestine  to  a 
virulent  organism  which  in  every  other  respect  is  identical. 
In  this  case  opinion  is  divided,  some  authorities  regarding  the 
toxic  or  pathogenic  B.  coli  as  a  distinct  species  from  the  non- 
toxic  B.  coli  although  they  possess  no  other  distinguishing 
feature  apart  from  virulence  and  this  in  the  case  of  the 
former  can  be  diminished  by  artificial  culture  and  in  the  case 
of  the  latter  increased  by  "  passage." 

The  discovery  of  the  amoeba  coli  in  the  intestines  of 
healthy  individuals  in  the  tropics,  where  amoebic  dysentery 
is  rife,  presents  a  similar  problem  for  solution.  Bahr  (1912) 
found  the  amoeba  coli  in  30  per  cent,  of  Fijians  examined  by 
him  and  Ash  burn  and  Craig  discovered  it  in  72  out  of  100 
soldiers  examined  in  the  Philippines  though  none  of  these 
72  had  diarrhoea  or  dysentery  at  the  time  or  had  ever  been 
on  the  sick  list  with  either  of  these  diseases. 

The  Bac.  anthracoides,  discovered  by  Andrewes  and  de- 
scribed by  Bainbridge  (1903),  was  distinguished  from  the  true 
B.  anthracis  by  slight  differences  in  the  appearance  of  gelatin 
and  agar  cultures,  in  rate  of  growth  and  in  motility  and  by 
its  non-virulence.  The  observations  of  Savage  and  MacConkey, 
already  quoted,  as  to  the  frequency  with  which  atypical 
colonies  of  some  organisms  occur  on  gelatin,  shows  how 


CH.  vi]  VARIATIONS  IN  VIRULENCE  93 

misleading  such  a  distinction  may  be,  and  in  this  case,  more- 
over, the  difference  in  the  appearance  of  colonies  on  agar 
could  by  the  adoption  of  certain  precautions  be  entirely 
eliminated.  The  rate  of  growth  of  organisms  is  always  subject 
to  variation.  Slight  motility  was  therefore  the  only  distin- 
guishing feature  to  which  any  importance  could  be  attached, 
apart  from  the  question  of  virulence.  As  regards  the  latter, 
experiments  indicated  that  this  property  could  be  increased 
by  passage.  Nevertheless  these  observers  regarded  the  dif- 
ference in  virulence  as  sufficiently  fundamental  to  justify 
their  description  of  the  organism  as  a  new  species  quite  dis- 
tinct from  the  B.  anthracis,  although  spores  of  the  latter 
organism  were  found  with  it. 

The  Streptococcus  erysipelatis  was  formerly  considered,  on 
account  of  its  greater  virulence  and  certain  minor  differences, 
to  be  a  distinct  species  from  S.  pyogenes\  further  investi- 
gation has  however  shown  this  opinion  to  be  untenable. 

Many  other  examples  might  be  given  but  these  will  suffice 
to  show  the  difficulties  that  arise  from  regarding  virulence  as 
a  "  specific  "  character. 


CHAPTER  VII 

VARIATIONS  IN  PATHOaENICITY 

WE  have  shown  that  morphology,  fermenting  properties  and 
virulence  are  all  variable  features.  There  remains  to  be 
considered  one  other  character  of  bacteria  which  is  of  great 
value  in  their  classification,  namely  their  "  pathogenicity  "  or 
their  power  to  cause  specific  disease. 

Under  this  head  are  to  be  considered,  firstly,  the  kind  of 
animal  in  which  a  particular  organism  can  develop  disease, 
secondly,  the  kind  of  symptoms  caused,  and  thirdly,  the  kind 
of  lesions  produced  by  that  organism's  invasion  of  the  living 
body. 

The  pathogenicity  of  an  organism  is  something  quite 
distinct  from  its  other  characters.  Two  organisms  may  possess 
the  same  morphology,  the  same  fermenting  power  and  the 
same  degree  of  virulence  and  yet  show  a  wide  divergence  in 
their  pathogenicity,  giving  rise  in  the  body  to  a  totally 
different  train  of  symptoms  and  lesions. 

This  character  of  pathogenicity  derives  particular  value 
and  importance  from  the  fact  that  it  is  generally  regarded  as 
being  more  "  fixed  "  than  the  other  characters  we  have  been 
discussing.  Great  reliance  is,  for  this  reason,  placed  upon 
resemblances  and  differences  in  pathogenicity  in  determining 
whether  two  organisms  do  or  do  not  belong  to  the  same 
species, — in  fact  it  is  regarded  as  constituting  a  final  appeal 
in  doubtful  cases. 

For  example,  Clark  (1910)  maintains  that  Hofmann's 
bacillus  and  the  Klebs-Loeffler  bacillus  represent  different 
species  on  the  ground  that  the  former  when  rendered  virulent 
gives  rise  to  different  symptoms  in  the  body. 

Again,  the  gonococcus  and  the  meningococcus  show  close 
resemblances  in  many  of  their  characters  but  are  readily 


CH.  vn]      VARIATIONS  IN  PATHOGENICITY  95 

distinguished  by  their  pathogenicity.  W.  B.  M.  Martin  (1911) 
writes  in  this  connection  :  "  so  far,  in  spite  of  the  prevalence 
of  gonorrhoea  and  the  periodical  occurrence  of  great  epidemics 
of  cerebrospinal  fever,  there  is  no  satisfactory  evidence  that 
the  gonococcus  ever  causes  a  meningitis  or  the  meningococcus 
a  urethritis.  This  is  the  more  remarkable  in  that,  on  the  one 
hand,  gonorrhoeal  metastases  are  common  enough  elsewhere 
and  that,  on  the  other,  meningococci  can  frequently  be  isolated 
from  the  urine  of  cases  in  which  there  is  not  the  slightest 
evidence  of  genito-urinary  inflammation."  He  maintains  that 
the  explanation  must  be  in  differences  in  "  pathogenicity  "  on 
the  part  of  the  two  organisms  and  that  such  differences  justify 
our  regarding  them  as  distinct  species. 

A  study,  however,  of  the  pathogenicity  of  bacteria  reveals 
the  fact  that  this,  like  every  other  character  they  possess,  is 
subject  to  variation — with  respect  to  (i)  the  kind  of  animal 
affected,  (ii)  the  kind  of  symptoms  caused  and  (iii)  the  kind 
of  lesions  produced. 

I.  As  regards  the  first  of  these  we  have  already  shown 
when  speaking  of  virulence  that  the  degree  to  which  a 
particular  organism  can  cause  disease  in  different  species  of 
animals  is  subject  to  variation  and  can  be  artificially  modified 
(vide  p.  81  et  seq.). 

II.  With  regard  to  the  second,  variability  in  the  symptoms 
caused  is  displayed  in  several  ways. 

1.  In  the  first  place,  the  same  species  of  organism  may 
give  rise  in  different  cases  to  a  totally  different  train  of 
symptoms. 

In  many  instances  the  explanation  is  obvious.  The  symptoms 
naturally  depend,  to  some  extent,  upon  the  particular  organ, 
either  primarily  or  solely,  affected  in  each  case.  The  toxic 
action  of  lead  furnishes  an  analogy.  Its  absorption  into  the 
body  is  followed  by  symptoms  of  arterio-sclerosis  or  of 
peripheral  neuritis  or  of  renal  disease  according  to  whether 
the  blood-vessels  or  the  nerves  or  the  kidneys  are  primarily 
affected.  The  pneumococcus,  for  example,  may  attack  the 
meninges,  the  lungs,  the  pericardium,  the  peritoneum  or  a 
synovial  membrane,  and  the  difference  in  the  symptoms  in 


96  VARIATIONS  IN  PATHOGENICITY      [CH.  vn 

each  case  is  attributable  to  anatomical  differences  in  the 
parts  affected. 

The  determining  factor  may  be  in  one  case  the  route  of 
infection,  in  another  the  lowered  vitality  of  the  particular 
organ  attacked.  In  a  third  case  neither  explanation  appears 
adequate  and  we  are  forced  to  conclude  that  the  organism 
itself  has  some  influence  in  determining  the  site  of  the 
disease. 

Such  an  hypothesis  is  required,  for  example,  to  explain 
the  fact  that  different  strains  of  the  tubercle  bacillus,  morpho- 
logically and  culturally  indistinguishable  from  one  another, 
may  produce  in  one  patient  phthisis,  in  another  a  tuberculous 
osteitis  or  arthritis,  and  in  a  third  lupus.  Here  the  analogy 
with  lead  poisoning  or  pneumococcal  infection  fails,  for  in 
both  the  latter  conditions,  although  one  system  or  organ  may 
be  for  a  time  affected  almost  alone,  the  disease  shows  a 
tendency  to  extend  to  other  regions  of  the  body  and  to 
produce  characteristic  symptoms  as  each  fresh  region  becomes 
involved.  In  the  case  of  tuberculous  infection  there  appear 
to  be  certain  limitations.  A  patient  with  phthisis  may  develop 
meningitis  or  peritonitis  or  general  tuberculosis,  but  it  is  rare 
for  a  phthisical  patient  to  develop  lupus  or  a  tuberculous 
joint,  or  for  one  suffering  from  a  tuberculous  ostitis  to  develop 
either  lupus  or  phthisis — facts  which  are  significant  when  one 
considers  how  widespread  are  these  diseases. 

Nield  and  Dunkley  (1909)  quote  an  instance  of  a  phthisical 
patient  who  moistened  a  scratch  on  her  arm  with  her  own 
saliva  and  developed  lupus  at  that  spot.  Examples  have  been 
given  by  various  observers  (quoted  Stel wagon,  1910)  of  lupus 
occurring  on  the  hands  of  women  employed  in  washing  the 
clothes  of  patients  with  phthisis  and  of  the  same  disease 
following  such  operations  as  ear  piercing,  tatooing  and  cir- 
cumcision when  performed  by  operators  themselves  suffering 
from  phthisis,  but  such  instances  are  sufficiently  uncommon 
to  be  regarded  as  exceptional. 

The  contrast  already  referred  to  between  the  gonococcus 
and  the  meningococcus,  in  respect  to  the  organs  they  par- 
ticularly— one  might  almost  say  exclusively — attack  and  the 


CH.  vn]     VARIATIONS  IN  PATHOGENICITY  97 

lesions  to  which  they  give  rise,  is  no  greater  than  the  contrast 
existing  between  different  strains  of  Koch's  bacillus  in  the 
same  respect ;  if  we  ascribe  the  contrast  in  the  former 
case  to  specific  differences  in  pathogenicity  one  is  forced  to 
ascribe  it  in  the  latter  case  to  the  same  factor  and  acknowledge 
that  different  strains  of  the  tubercle  bacillus  exhibit  marked 
differences  in  pathogenicity. 

There  is  one  not  unlikely  fallacy  which  needs  to  be 
guarded  against  before  we  can  with  confidence  attribute  to 
an  organism  any  unusual  symptoms  which  appear  to  follow 
its  invasion  of  the  body.  A  certain  disease  may  be  latent  in 
the  patient,  that  is  to  say  present  without  giving  rise  to  any 
noticeable  symptoms.  The  constitutional  disturbances  arising 
from  infection  by  the  organism  in  question  may  "  light  up  " 
this  pre-existing  disease  and  the  symptoms  of  the  latter  may 
then  be  incorrectly  credited  to  the  invading  organism. 

Such  a  sequence  is  well  illustrated  by  a  case  recorded  by 
Roberts  and  Ford  under  the  title  "  A  case  of  Cerebrospinal 
Fever  simulating  Acute  Nephritis  with  uraemic  convulsions." 
The  patient  suffered  from  typical  symptoms  of  acute  nephritis 
and  uraemia — dropsy,  the  passage  of  scanty  urine  loaded  with 
albumen,  convulsions  and  coma — and  showed  marked  im- 
provement as  a  result  of  the  treatment  usually  adopted  for 
these  conditions.  The  meningococcus  was,  however,  isolated 
from  the  spinal  fluid  and  the  symptoms  on  this  account 
attributed  to  that  organism.  In  this  case  a  pre-existing 
nephritis  might,  conceivably,  have  given  rise  at  the  onset  of 
the  illness  to  the  symptoms  characteristic  of  that  disease 
before  those  characteristic  of  cerebrospinal  fever  had  had 
time  to  develop  ;  or  the  symptoms  of  the  first  disease  might 
have  completely  masked  those  of  the  second. 

2.  In  the  second  place,  one  and  the  same  strain  of  an 
organism  can  by  artificial  means  be  so  modified  as  to  cause 
an  altogether  different  type  of  disease.  For  example,  Madame 
Henri  (1914)  found  that  the  pathogenicity  of  B.  anthracis 
was  changed  to  a  remarkable  degree  by  exposure  to  the 
ultra-violet  rays.  Its  subsequent  injection  into  an  animal 
produced  symptoms  quite  unlike  those  caused  by  the  normal 
D.  7 


98  VARIATIONS  IN  PATHOGENICITY     [OH.  vn 

anthrax  bacillus  and  it  did  not  revert  after  daily  subculture 
for  two  months  afterwards. 

3.  In  the  third  place,  a  contagious  disease  passed  from 
one  case  to  another  during  the  course  of  an  epidemic  may  be 
characterised  in  different  cases  by  widely  different  symptoms. 

Andre wes  and  Horder  (1906)  have  recorded  an  example 
of  this.  A  woman  (A),  admitted  for  her  confinement  to  a 
maternity  home,  was  attended  by  a  nurse  (B)  who  developed 
tonsilitis  on  the  day  the  patient  was  delivered,  and  three 
days  later  was  notified  as  a  case  of  scarlet  fever.  The  woman 
(A)  developed  puerperal  fever  and  was  removed  to  a  hospital 
where  she  died.  Three  days  after  her  death  a  nurse  (C)  who 
had  attended  her  at  the  hospital  developed  scarlet  fever.  At 
the  maternity  home  another  nurse  (D)  took  nurse  B's  place 
and  had  attended  the  woman  (A)  for  a  day  or  two  before  the 
latter's  removal  to  hospital.  Ten  days  later  this  nurse  (D), 
who  herself  remained  well,  attended  another  confinement  case 
(E)  in  the  district.  The  woman  (E)  died  of  septicaemia  on 
the  fifth  day  after  delivery.  Nurse  B's  room,  having  been 
disinfected  by  the  Sanitary  Authority,  was  scrubbed  by  a 
charwoman  (F).  On  the  following  day  this  charwoman  became 
ill  but  the  case  was  not  recognised  to  be  scarlet  fever  until  a 
day  or  two  later  when  her  two  children  (G)  and  (H)  developed 
typical  scarlet  fever  and  all  three  were  removed  to  a  fever 
hospital.  A  scheme  will  make  the  sequence  of  events  clearer : 

^  Child  (G) 
Maternity  nurse  (B)  Charwoman  (F)         /      Scarlet  fever 


Scarlet  fever  Scarlet  fever  \      Child  (H) 

Scarlet  fever 

Patient  (A)  ^  Maternity  nurse  (D)  — >•  District  case  (E) 

Puerperal  fever  well  Puerperal  fever 


Hospital  nurse  (C) 
Scarlet  fever 

The  original  case  of  scarlet  fever  infected  two  other  people, 
one  with  scarlet  fever  and  the  other  with  puerperal  fever ; 
this  case  of  puerperal  fever  also  infected  two  other  people, 
one  with  puerperal  fever  and  the  other  with  scarlet  fever. 

An  even  more  remarkable  instance  is  recorded  by  Dunn 


CH.  vn]      VARIATIONS  IN  PATHOGENICITY  99 

and  Gordon  (1905).  They  describe  an  epidemic  in  Hertford- 
shire characterised  by  an  extraordinary  diversity  of  symptoms 
in  different  patients.  In  some  cases  there  were  sneezing, 
coryza,  and  the  ordinary  symptoms  of  a  common  cold.  In 
other  cases  patients  had  "aches  and  pains  all  over,"  stiff  neck 
and  suffered  subsequently  from  great  debility ;  such  cases 
had  all  the  appearances  of  influenza.  In  others,  again,  the 
illness  closely  simulated  scarlet  fever ;  it  began  with  sore 
throat,  rigors,  vomiting,  headache,  fever  and  rapid  pulse,  and 
was  accompanied  by  a  punctate  rash  at  the  end  of  the  first 
24  hours  (followed  later  by  desquamation),  the  "  strawberry  " 
tongue,  circumoral  pallor,  enlarged  cervical  glands  which  in 
some  cases  suppurated,  and,  in  some  patients,  complications 
such  as  nephritis,  arthritis  and  otorrhoea.  A  fourth  type 
resembled  diphtheria  and  exhibited  a  suspicious  membrane 
on  the  tonsil.  A  fifth  type  was  notified  in  some  cases  as  typhoid 
fever  and  was  characterised  by  epistaxis,  melaena,  prostra- 
tion and,  in  some  cases  it  is  stated,  a  positive  Widal  reaction. 
Finally,  a  number  of  cases,  particularly  amongst  children, 
resembled  cerebrospinal  fever  and  were  so  diagnosed ;  these 
were  characterised  by  profuse  nasal  discharge,  pain  in  the 
back  of  the  neck,  headache,  photophobia  and  irritability, 
dilatation  of  one  or  both  pupils,  persistent  vomiting,  drowsi- 
ness, head  retraction,  paralysis,  coma  and,  sometimes,  con- 
vulsions and  death. 

Sometimes  these  widely  divergent  types  were  exhibited 
by  the  different  members  of  a  single  family  or  household 
struck  down  by  the  disease,  either  simultaneously  or  con- 
secutively. After  a  thorough  investigation  these  observers 
were  convinced  that  the  outbreak  of  these  various  types  of 
illness  was  due  to  the  prevalence  and  spread  of  only  one 
disease  and  not  a  number  of  different  diseases,  and  a  bacterio- 
logical examination  of  a  large  number  of  cases  by  Gordon 
showed  that  the  disease  was  due  to  infection  by  an  organism 
closely  resembling,  if  not  identical  with,  M .  catarrhalis. 

4.  In  the  fourth  place,  the  same  species  of  organism  may 
give  rise  in  different  epidemics  to  widely  different  types  of 
disease.  For  example,  strains  of  B.  influenzae  may  give  rise 

7—2 


100  VARIATIONS  IN  PATHOGENICITY     [CH.  vn 

to  epidemics  of  "influenza"  characterised  by  symptoms 
resembling  in  one  epidemic  a  simple  coryza,  in  another 
rheumatic  fever,  in  a  third  typhoid  fever,  and  in  a  fourth 
cerebrospinal  meningitis. 

5.  In  the  fifth  place,  the  train  of  symptoms  characteristic 
of  infection  by  one  organism  may  develop  as  a  result  of 
infection  by  a  totally  different  organism. 

A  striking  instance  of  this  is  recorded  by  Head  and 
Wilson  (1899)  who  proved  that  a  supposed  case  of  rabies  was 
actually  due  to  infection  by  the  diphtheria  bacillus.  The 
diagnosis  of  rabies  was  founded  on  the  history  and  clinical 
symptoms.  "  The  well  authenticated  history  of  a  bite  on  the 
cheek  by  an  unknown  animal,  the  two  months'  incubation 
period,  the  onset  with  extreme  pain  and  numbness  in  the 
region  of  the  scar,  the  development  of  the  characteristic 
laryngeal  and  respiratory  spasms  on  attempting  to  take 
liquids,  the  spasm  at  first  being  slight  but  later  more  pro- 
nounced and  towards  the  close  again  feeble  or  absent,  the 
insomnia,  the  absence  in  the  beginning  of  fever  which  later 
in  the  illness  became  pronounced,  the  rapid  pulse  at  all 
stages,  the  attacks  of  violent  delirium  interspersed  with 
periods  of  calm  and  complete  rationality,  the  absence  of  all 
symptoms  pointing  towards  any  other  simulating  disease  and 
the  fatal  termination — all  serve  to  make  an  almost  complete 
picture  of  rabies."  The  Klebs-Loeffler  bacillus  was  isolated 
from  the  ventricular  fluid  and  detected  in  the  nerve  cells  of 
the  medulla.  The  recognition  of  this  organism  was  complete 
and  beyond  doubt.  "  Not  less  suggestive  of  rabies  than  the 
clinical  history  were  the  results  of  subdural  inoculations  of 
rabbits  with  emulsions  prepared  from  the  medulla  of  the 
patient.  There  occurred  the  long  period  of  incubation  (20 
and  21  days)  followed  by  phenomena  similar  to  those  in 
experimental  rabies  of  rabbits,  and  other  rabbits  inoculated 
subdurally  with  the  medulla  of  the  first  rabbits  behaved  in 
a  similar  manner."  B.  diphtheriae  was  demonstrated  after 
death  in  the  medulla  of  the  rabbits.  By  a  thorough  investiga- 
tion, full  details  of  which  are  given,  infection  by  the  virus  of 
rabies  was  definitely  excluded. 


CH.  vii]     VARIATIONS  IN  PATHOGENICITY  101 

Dunn  and  Gordon  (1905,  vide  supra  p.  99)  have  described 
almost  typical  cases  of  scarlet  fever,  of  cerebrospinal 
fever  and  of  influenza,  which  proved  to  be  due  to  infec- 
tion by  the  micrococcus  catarrhalis.  Gordon  has  described 
elsewhere  typical  cases  of  cerebrospinal  fever  due  to  B. 
typliosm. 

Nash  has  recorded  a  remarkable  case  of  malignant  en- 
docarditis characterised  by  fever,  constipation,  headache, 
drowsiness  and  delirium,  photophobia,  strabismus,  head  re- 
traction and  the  appearance  of  a  petechial  rash.  The  illness, 
in  fact,  presented  all  the  clinical  features  of  cerebrospinal 
fever.  A  copious  growth  of  a  pure  culture  of  the  Klebs- 
Loeffler  bacillus  was  obtained  post  mortem  from  the  spinal 
fluid  and  a  similar  growth  from  the  heart's  blood.  There  was 
a  history  of  a  discharge  from  the  ear  at  the  beginning  of  the 
illness  but  no  history  of  sore  throat. 

Thomson  (1911)  has  recorded  his  own  experience  of  an 
acute  inflammation  of  the  throat  simulating  diphtheria  in 
producing,  in  the  fourth  week  of  the  illness,  temporary  para- 
lysis of  the  tongue,  arms  and  legs,  but  proved  to  be  due  to 
pneumococcal  infection. 

Colman  and  Hastings  (1909)  state  their  conviction  that 
some  strains  of  B.  coli  are  capable  of  causing  a  disease  clini- 
cally identical  with  typhoid  fever. 

III.  The  pathogenicity  of  bacteria  presents  yet  another 
aspect,  namely  the  character  of  the  lesions  produced  by  them 
in  the  living  tissues. 

This  can  be  studied  in  two  ways.  Firstly,  by  observing  the 
lesions  produced  in  the  body  at  various  stages  in  the  course 
of  an  infective  disease  ;  and  secondly,  by  observing  the  lesions 
produced  by  the  artificial  inoculation  of  organisms  into 
animals,  both  at  the  site  of  inoculation  and  elsewhere. 

1.  The  lesions  produced  in  the  course  of  disease  and  ob- 
served post  mortem  not  infrequently  enable  one  to  identify 
the  infecting  organism.  For  example,  tuberculous  ulceration 
of  the  intestine,  tuberculous  consolidation  of  the  lungs,  and 
tuberculous  invasion  of  the  skin,  present  altogether  different 
features  from  typhoid  ulceration  of  the  intestine,  pneumococcal 


102  VARIATIONS  IN  PATHOGENICITY     [OH.  vn 

consolidation  of  the  lung  and  streptococcal  invasion  of  the 
skin,  respectively. 

It  is  however  common  experience  that  even  in  the  post 
mortem  room  a  certain  diagnosis  of  the  nature  of  the  infec- 
tion cannot  always  be  made.  Sydney  Martin,  in  speaking  of 
tuberculosis,  says  "There  is,  with  the  exception  of  the  presence 
of  the  tubercle  bacillus,  no  element  in  the  structure  of  the  tu- 
berculous lesion  which  is  diagnostic  of  the  disease."  In  other 
words  the  lesions  regarded  as  characteristic  of  infection  by 
one  species  of  organism  may  be  produced  by  infection  by  a 
totally  different  species. 

Such  departures  from  what  experience  has  taught  us  to 
regard  as  the  normal  or  characteristic  lesion  in  the  case  of  a 
given  organism  may  be  accounted  for  by  the  influence  of  other 
factors  beside  the  nature  of  the  organism  itself— such  factors, 
for  example,  as  the  age  of  the  patient,  the  route  of  invasion, 
the  presence  of  a  secondary  infection,  the  effect  of  treatment, 
and  many  others.  The  question  arises,  how  far,  if  it  were 
possible  to  exclude  such  disturbing  influences,  would  the 
lesions  retain  their  specific  character  ? 

2.  This  leads  us  to  a  consideration  of  the  second  method 
of  studying  the  question — by  observing  the  lesions  produced 
by  artificial  inoculation  of  animals,  both  at  the  site  of  inocu- 
lation and  elsewhere.  Such  a  method  enables  one  to,  so  to 
speak,  "standardise"  the  lesion.  A  healthy  animal  of  the 
same  species,  age  and  weight  can  be  utilised  at  each  experi- 
ment, the  inoculation  made  in  the  same  manner,  at  the  same 
site,  with  the  same  number  of  organisms  and  these  of  the  same 
degree  of  virulence,  and  the  animal  can  be  killed  after  the 
same  interval  of  time. 

Many  investigators  maintain  that  under  such  conditions 
the  lesions  produced  by  a  certain  species  of  organism  are 
constant  in  their  appearance — that,  however  much  the  other 
characters  of  an  organism  may  vary,  this  character  at  any 
rate  is  invariable  and  will  establish  beyond  dispute  to  which 
of  two  species  a  doubtful  organism  actually  belongs. 

Thus,  Klein  as  long  ago  as  1899  in  describing  the  "bacillus 
of  pseudo-tuberculosis  "  stated  that  in  cultural  and  morpho- 


CH.  vn]      VARIATIONS  IN  PATHOGENICITY  103 

logical  characters  this  organism  showed  certain  resemblances 
to  B.  coli.  The  two  organisms  could  be  distinguished  from 
each  other  most  certainly  by  animal  inoculation.  Subcutaneous 
inoculation  of  the  first  named  into  the  guineapig  gave  rise  to 
typical  nodular,  necrotic,  purulent  changes  in  the  lymphatic 
glands,  omen  turn,  pancreas,  liver,  spleen,  and  lung,  an  effect 
which  B.  coli  and  its  varieties  did  not  produce. 

Again,  Shattock  (and  others,  1907)  regards  the  avian  tu- 
bercle bacillus  and  the  human  tubercle  bacillus  as  two  distinct 
species  on  the  ground  that,  whereas  the  former  when  inoculated 
into  guineapigs  produces  merely  a  local  or  a  local  and  glandu- 
lar disease,  the  latter  produces  visceral  disease  as  well. 

Savage  (1908-9)  has  recorded  some  interesting  experiments 
illustrating  the  value  of  animal  inoculation  in  revealing  differ- 
ences in  pathogenicity.  He  found  that  streptococcus  mastitidis, 
which  causes  mastitis  in  the  cow,  was  non-virulent  to  mice 
and  other  rodents  but  possessed  to  a  marked  degree  the  power 
to  produce  mastitis  in  goats  when  inoculated  into  the  mammary 
ducts,  and  was  thereby  differentiated  from  streptococcus  an- 
ginosa  (isolated  from  human  sore  throat)  which,  though  virulent 
to  mice,  did  not  possess  the  power  to  produce  mastitis  in  goats. 
Continuing  his  experiments  with  pyogenic  streptococci  derived 
from  many  sources,  he  found  that,  although  in  their  cultural 
properties  and  their  virulence  to  mice  they  displayed  wide 
differences,  they  all  resembled  each  other  in  their  inability  to 
produce  mastitis  in  goats.  One  streptococcus,  for  example, 
isolated  from  a  fatal  lymphadenitis  in  a  boy,  after  it  was  in- 
oculated into  the  teat  of  a  goat  survived  for  seven  months  as 
a  harmless  saprophyte  in  the  milk  passages. 

One  other  example  will  suffice.  We  recognise  clinically 
two  types  of  pneumonia,  lobar  or  croupous  pneumonia  and 
lobular,  catarrhal  or  broncho-pneumonia.  Both  types  may 
result  from  infection  of  the  lung  by  the  pneumococcus.  The 
invading  organism  is  apparently  identical  in  the  two  cases, 
judged  by  the  ordinary  cultural  and  morphological  tests,  and 
the  difference  in  the  results  produced  are  therefore  attributed 
to  differences  in  the  age  and  vitality  of  the  patient  and  the 
route  of  infection. 


104  VARIATIONS  IN  PATHOGENICITY     [CH.  vn 

Eyre,  Leatham  and  Washbourne  (1906)  endeavoured  by 
the  method  of  animal  inoculation  to  ascertain  whether  the 
difference  in  the  lesions  caused  depended  upon  specific  differ- 
ences in  the  pathogenicity  of  the  infecting  strains.  They 
found  that  strains  of  the  pneumococcus  isolated  from  cases  of 
lobar  pneumonia  when  inoculated  subcutaneously  into  the 
guineapig  almost  invariably  gave  rise  to  a  local  inflammatory 
exudation  of  a  fibrinous  type,  whereas  strains  isolated  from 
cases  of  broncho-pneumonia,  when  similarly  inoculated,  almost 
invariably  gave  rise  to  a  local  inflammatory  exudation  of  a 
celMar  type,  easily  distinguished  from  the  other.  A  number 
of  strains  of  pneumococci  obtained  from  a  "  neutral "  source, 
such  as  the  mouth,  likewise  showed  differences  in  the  nature 
of  the  inflammatory  reaction  they  provoked  at  the  site  of  in- 
oculation, some  belonging  to  the  "fibrinous"  type  and  others 
to  the  "  cellular  "  type.  They  further  showed  that  this  feature 
was  not  associated  with  any  other  differences  between  the 
strains  as  regards  morphology  or  cultural  characters  or  fer- 
menting properties  and  was  quite  independent  of  their  degree 
of  virulence.  They  therefore  regarded  it  as  a  specific  character. 

If  the  lesions  produced  in  the  body  during  the  course  of 
an  infective  disease  are  subject  to  variation,  are  those  which 
result  from  the  artificial  inoculation  of  animals  any  more 
constant? 

The  materials  from  which  to  form  an  opinion  on  this  point 
are  somewhat  scanty.  That  the  feature  in  some  cases  is  very 
constant  was  shown  by  Shattock  (and  others,  1907)  by  means 
of  the  following  experiment.  They  grew  a  strain  of  human 
tubercle  bacilli  for  eight  weeks  in  the  spleen  of  a  pigeon.  The 
subsequent  inoculation  of  the  organisms  into  a  guineapig  gave 
rise,  not  as  might  have  been  expected  to  the  lesions  charac- 
teristic of  avian  tubercle,  but  to  those  characteristic  of  the 
human  type.  Baldwin  (1910)  likewise  grew  the  human  type 
of  tubercle  bacillus  for  19  months  continuously  in  the  bovine 
tissues  without  in  any  way  affecting  its  pathogenic  powers  to- 
wards rabbits  and  guineapigs. 

On  the  other  hand,  we  have  quoted  in  an  earlier  paragraph 
an  instance  of  a  certain  strain  of  the  diphtheria  bacillus  which 


CH.  vn]      VARIATIONS  IN  PATHOGENICITY  105 

not  only  gave  rise  to  atypical  symptoms  and  lesions  (namely 
those  of  rabies)  in  the  human  body  in  the  course  of  disease 
but  produced  no  less  atypical  lesions  when  inoculated  into  a 
rabbit  (vide  p.  100). 

Again,  Savage  (1908-9)  found  in  further  experiments  that 
a  virulent  strain  of  the  streptococcus  mastitidis  from  the  udder 
secretion  in  a  case  of  bovine  mastitis,  under  certain  conditions 
(namely  3  days'  residence  in  the  human  pharynx),  was  almost 
deprived  of  its  characteristic  power  to  produce  mastitis  in 
goats. 

Again,  Mohler  and  Washburn  (1906)  claim  that  the  various 
types  of  tubercle  bacilli — human,  bovine,  avian — can  be  readily 
converted  one  into  another,  by  prolonged  residence  in  a  suitable 
animal  host,  so  as  to  be  indistinguishable  by  the  ordinary  in- 
oculation tests. 

Rosenow  (1914)  obtained  a  strain  of  haemolysing  strepto- 
cocci from  the  throat  in  a  case  of  scarlet  fever.  A  culture  on 
blood  agar  yielded  two  distinct  kinds  of  colonies,  (a)  non-ad- 
herent colonies  of  a  haemolysing  organism  which  fermented 
mannite  but  failed  to  ferment  maltose  and  saccharose, 
(6)  adherent,  green-producing  colonies  of  a  non-haemolysing 
organism  which  would  not  ferment  mannite  but  fermented 
maltose  and  saccharose.  When  injected  into  a  rabbit,  the 
former  attacked  primarily  the  joints  while  the  latter  showed 
a  predilection  for  the  heart  valves.  In  other  words,  the  original 
strain  on  artificial  cultivation  gave  rise  to  two  strains  which 
differed  in  their  pathogenicity. 

Finally,  may  be  mentioned  Foa's  experiments  (1890).  He 
inoculated  a  rabbit  with  the  diplococcus  lanceolatus  capsulatus 
with  a  fatal  result.  From  this  dead  rabbit  he  inoculated  two 
others,  the  first  by  injecting  organisms  derived  from  some  of 
the  fresh  fibrinous  pneumonic  exudate  in  the  lung,  and  the 
second  by  injecting  organisms  derived  from  the  cerebrospinal 
fluid.  He  found  that  the  disease  set  up  in  these  two  rabbits 
differed.  The  first  rabbit  showed,  for  example,  an  inflammatory 
oedema  of  the  skin ;  the  second  did  not  show  this.  He  found, 
however,  that  if  the  strain  isolated  from  the  lung  were  grown 
anaerobically  and  then  injected  into  a  rabbit  the  effects  it 


106  VARIATIONS  EST  PATHOGENICITY      [OH.  vn 

produced  were  indistinguishable  from  those  produced  by  the 
strain  isolated  from  the  spinal  fluid. 

Whatever  aspect  of  pathogenicity,  therefore,  we  study,  the 
same  feature  becomes  apparent — namely,  that  this  property 
of  bacteria  is,  like  others,  subject  to  variation. 

VARIATION  IN  OTHER  CHARACTERS  OF  BACTERIA. 

In  the  foregoing  pages  variations  in  morphology,  ferment- 
ing power,  virulence  and  pathogenesis  have  been  discussed  in 
detail.  There  remain  many  more  characters  of  bacteria  to  be 
considered — such  as  their  viability,  their  staining  properties, 
their  power  to  produce  indol  and  to  liquefy  gelatin,  their  ag- 
glutination reactions  and  many  others.  It  would  be  easy 
to  illustrate  the  variations  these  characters  also  undergo 
under  different  conditions.  Many  examples  will  be  found  in 
Chapter  II. 


CHAPTER  VIII 

THE  POSSIBLE  OCCURRENCE  OF  TRANSMUTATION 
IN  THE  LIVING  BODY 

THE  significance  of  the  variations  recorded  in  the  foregoing 
sections,  with  reference  to  the  question  whether  actual  trans- 
mutation of  bacteria  can  be  brought  about  artificially  or  not, 
will  be  dealt  with  later.  It  is  proposed,  at  this  point,  to  consider 
another  aspect  of  the  problem,  namely  the  possibility  of 
transmutation  occurring  in  the  tissues  of  the  living  body. 

In  certain  regions  of  the  body  one  finds  growing  side  by 
side  two  strains  of  organisms  closely  resembling  each  other 
in  every  respect  save  one — namely  their  pathogenicity.  One 
strain  is  capable  of  causing  a  definite  train  of  lesions  and 
symptoms ;  the  other,  as  a  rule,  does  not  give  rise  to  any  signs 
of  disease.  The  suggestion  that  one  strain  may  be  in  some  way 
a  derivative  of  the  other  offers  a  tempting  hypothesis  to  explain 
both  their  resemblance  and  their  proximity  to  each  other. 
An  illustration  will,  perhaps,  make  this  clearer.  In  the  hides 
of  cattle  may  sometimes  be  found  non-virulent  bacilli  closely 
resembling  B.  anthracis.  Such  an  organism  was  discovered 
by  Andre wes  and  described  by  Bainbridge  (1903)  under  the 
name  B.  anthracoides  (vide  p.  92).  The  organism  was 
stated  to  differ  from  B.  anthracis  in  the  appearance  of  its 
colonies,  in  its  rate  of  growth,  in  possessing  slight  motility  and 
in  being  non-virulent.  By  slightly  modifying  the  conditions  of 
growth,  colonies  on  agar  could  be  made  to  assume  the  typical 
appearance  of  anthrax  colonies,  while  its  virulence  proved 
capable  of  increase  by  "passage."  The  differences  in  character 
between  this  organism  and  B.  anthracis  were  deemed  sufficient 
by  these  observers  to  justify  them  in  classifying  it  as  a  distinct 
species,  but  it  is  difficult  to  resist  the  conclusion  either  that 


108  THE  POSSIBLE  OCCURRENCE  OF     [OH.  vm 

the  non-virulent  organism  was  a  direct  derivative  of  the  true 
anthrax  bacillus  or  that  it  would  be  capable  of  giving  rise  to 
the  latter  under  suitable  conditions.  Such  a  supposition  is 
favoured,  firstly,  by  the  admission  that  the  bundle  of  horse  hair 
from  which  the  B.  anthracoides  was  isolated  contained  also 
the  spores  of  true  anthrax,  and,  secondly,  by  the  discovery  of 
Hueppe  and  Wood  some  years  before  (1889)  of  a  similar  non- 
virulent  saprophytic  anthrax-like  organism  in  earth,  which 
however  on  injection  into  a  mouse  rendered  the  animal  immune 
to  anthrax. 

Similar  examples  of  association  between  non-virulent  and 
virulent  organisms,  otherwise  closely  resembling  each  other, 
may  be  found  in  the  human  body — in  the  intestine  B.  coli  and 
B.  typhosus,  in  the  throat  Hofmann's  bacillus  and  the  Klebs- 
Loeffler  bacillus,  in  the  skin  the  Staphylococcus  epidermidis 
albus  and  the  Staphylococcus  pyogenes  aureus,  in  the  naso- 
pharynx the  micrococcus  catarrhalis  and  the  meningococcus. 

The  exact  relationship  in  each  case  has  never  been  satis- 
factorily determined.  Over  twenty  years  ago  Adami  (quoted 
by  Arloing,  1891)  put  forward  the  suggestion  that  B.  coli  might 
give  rise  in  the  presence  of  fermenting  faecal  matter  to 
B.  typhosus,  a  theory  which  has  been  recently  revived  by 
Tarchette  (1904)  and  others  (quoted  by  Hamer,  1909). 

The  precise  relationship  between  the  virulent  Klebs-Loeffler 
bacillus  and  Hofmanns  bacillus  is  still  a  matter  of  controversy. 
The  latter  is  a  harmless  saprophyte  not  infrequently  found  in 
the  pharynx  of  healthy  persons.  It  is  distinguished  from  the 
true  diphtheria  bacillus  by  the  somewhat  different  appearance 
of  its  colonies  on  artificial  media,  by  slight  and,  according  to 
some  observers,  inconstant  differences  in  its  morphology  and 
staining,  by  its  inability  to  ferment  glucose  and  other  sugars, 
and  by  being  non-pathogenic  to  man  and  to  the  guineapig. 
It  has  not  been  found  possible  to  produce  immunity  against 
true  diphtheria  by  inoculation  with  Hofmann's  bacillus,  and 
the  injection  of  a  filtered  broth  culture  of  the  latter  does  not 
give  rise  to  antitoxin  formation  in  the  horse  (Petrie,  1905) 
though  the  filtrate  in  the  case  of  even  avirulent  Klebs-Loeffler 
bacilli  will  do  so  (Arkwright,  1909).  Nevertheless  many  in- 


CH.  vin]  TRANSMUTATION  IN  THE  LIVING  BODY  109 

vestigators  claim  to  have  converted  the  Klebs-Loeffler  type 
of  organism  into  the  Hofmann  type — by  prolonged  cultivation 
(Lesieur,  1901),  by  growth  at  a  high  temperature  (Hewlett  and 
Knight,  1897),  by  growth  in  the  subcutaneous  tissues  of  an 
immune  rat  (Ohlmacher,  1902)  and  other  methods — and  main- 
tain that  the  reverse  change  can  be  brought  about  by  "passage" 
(Lesieur,  1901,  Hewlett  and  Knight,  1897,  Ohlmacher,  1902, 
etc.).  Salter  (1899)  has  stated  that,  by  five  successive  passages 
through  goldfinches,  he  was  able  to  convert  four  strains  of 
typical  Hofmann's  bacilli  into  no  less  typical  Klebs-Loeffler 
bacilli,  the  transformation  being  complete  as  regards  virulence, 
morphology  and  acid  production,  and  in  the  power  to  form 
a  toxin  neutralised  by  diphtheria  antitoxin. 

Thiele  and  Embleton  claim  to  have  converted  Hofmann's 
bacillus  into  a  bacillus  morphologically  indistinguishable  from 
the  diphtheria  bacillus  and  capable  of  secreting  an  exotoxin 
which  can  be  neutralised  by  diphtheria  antitoxin.  This  was 
accomplished  by  inoculating  a  succession  of  guineapigs  with 
an  emulsion  of  Hofmann's  bacillus  containing  a  certain  pro- 
portion of  gelatin,  the  organism  being  recovered  from  the 
peritoneal  cavity  after  each  passage. 

As  regards  the  fermenting  properties  of  the  two  organisms, 
Clark  (1910)  has  shown  that  Hofmann's  bacillus  does  produce 
slight  acidity  in  dextrose  broth;  while  Goodman  (1908),  by  a 
process  of  selection,  obtained  strains  of  the  true  diphtheria 
bacillus  which  exhibited  differences  in  fermenting  power  as 
wide  as  those  naturally  existing  between  this  organism  and 
Hofmann's ;  and  he  concluded  that  the  fermenting  power  was 
a  poor  guide  in  determining  whether  an  organism  was  a 
pathogenic  one  or  a  harmless  saprophyte. 

Finally,  Boycott's  statistics  demonstrate  (Muir  and  Ritchie) 
that  the  period  of  maximal  seasonal  prevalence  of  Hofmann's 
bacillus  immediately  precedes  that  of  true  diphtheria,  and 
Hewlett  and  Knight  (1897)  have  offered  evidence  in  support 
of  the  opinion  that  Hofmann's  bacillus  is  present  in  increasing 
numbers  in  the  throats  of  diphtheria  patients  during  recovery 
from  the  disease. 

Recent  work  by  Graham  Smith  and  others,  and  the  inability 


110  THE  POSSIBLE  OCCURRENCE  OF    [CH.  vin 

of  these  observers,  on  repeating  the  experiments  of  earlier 
investigators,  to  obtain  the  same  results,  somewhat  invalidates 
the  conclusions  of  the  latter,  so  that  the  question  of  the  possi- 
bility of  a  mutation  between  the  two  species  remains  sub 
judice. 

Several  species  of  staphylococci  are  recognised, — £ 
epidermidis  albus,  S.  pyogenes  albus,  S.  pyogenes  aureus. 
The  distinction  between  these  three  rests  on  their  inequality 
in  virulence,  on  their  different  powers  of  fermenting  carbo- 
hydrates, and,  as  their  names  imply,  on  their  dissimilarity  in 
the  production  of  pigment. 

As  regards  virulence,  the  first-named  organism  is  normally 
present  in  the  skin  of  healthy  persons  and  is  non-pathogenic ; 
the  second  possesses  slight  virulence,  producing  mild  local 
inflammatory  conditions ;  while  the  third  is  a  virulent  organism 
found  in  pathological  conditions  such  as  suppurative  cutaneous 
and  subcutaneous  lesions,  acute  bone  infection  and  septicaemia. 
Staphylococcus  epidermidis  albus  may  however  assume  a 
certain  degree  of  virulence  and  give  rise  to  a  stitch  abscess 
or  mild  inflammation  (Dudgeon  and  Sargent,  1907)  and  plays 
an  important  role  in  peritonitis  (ibid.).  Andre wes  and  Gordon 
(1905-6)  isolated  it  in  pure  culture  in  one  case  of  otitis  media 
and  also  from  a  boil.  The  Staphylococcus  pyogenes  albus  can 
be  made  much  more  virulent  by  artificial  passage.  It  has  been 
known  to  become  parasitic,  invading  the  human  body  and 
circulating  in  the  blood  stream  (Panichi,  1906,  Southard,  1910). 

In  the  second  place,  as  regards  their  fermenting  properties, 
Gordon  (1904-5)  has  shown  that  strains  of  Staphylococcus 
albus  isolated  from  the  skin  of  healthy  persons  show  very 
great  diversity  in  their  fermenting  power.  In  an  earlier  paper 
(1903-4)  he  describes  two  strains,  one  a  Staph.  albus  derived 
from  the  skin  and  the  other  a  Staph.  pyogeiiies  aureus  derived 
from  pus — which,  when  "put  through"  no  less  than  20  carbo- 
hydrate substances,  revealed  different  fermenting  power  in 
one  only,  namely  mannite. 

In  the  third  place,  as  regards  pigment  formation,  it  has 
been  proved  by  many  investigators  (Neumann,  Dudgeon,  1908, 
Andrewes  and  Gordon,  1905-6)  that  non-pigmented  cocci  can 


CH.  vin]   TRANSMUTATION  IN  THE  LIVING  BODY  111 

be  obtained  on  culture  from  pigmented  ones,  and  that  cocci 
which  fail  to  produce  pigment  under  certain  conditions  will 
<lo  so  readily  if  the  conditions  are  altered  (vide  p.  15).  Dudgeon 
(1908)  cites  one  experiment  in  which  a  Staphylococcus  aureus 
was  injected  into  an  animal  and  a  Staphylococcus  albus  was 
recovered  from  the  spleen  at  its  death.  In  the  last  case 
Gordon's  tests  were  identical  in  both  instances,  showing  that 
the  character  of  pigment  production  was  the  only  one  to 
undergo  modification,  but  it  does  not  require  a  great  stretch 
of  imagination  to  suppose  that  just  as  the  virulent  parasitic 
pneumococcus  and  the  avirulent  saprophytic  variety  may 
undergo  mutation  (vide  p.  82)  so  the  highly  virulent  "aureus" 
and  the  less  virulent  "albus"  might  under  certain  circum- 
stances be  converted  the  one  into  the  other. 

Many  more  hypotheses  of  the  same  nature,  and  based  on 
similar  evidence,  might  be  put  forward  with  varying  degrees 
of  plausibility.  One  other  example  will  suffice,  namely  the 
question  of  the  relationship  of  the  meningococcus  to  two 
other  diplococci — Mic.  catarrhalis  on  the  one  hand  and  the 
pneumococcus  on  the  other. 

The  Micrococcus  catarrhalis  which  is  frequently  present 
in  the  mouths  of  healthy  persons,  especially  children,  is  an 
organism  resembling  in  many  respects  the  meningococcus,  but 
of  low  virulence.  The  two  organisms  are,  as  a  rule,  easily 
distinguished  by  important  differences  existing  between  them. 
Thus  the  meningococcus  is  much  smaller  than  Mic.  catarrh- 
alis: its  colonies  are  also  smaller  and  their  outlines  more 
regular ;  it  liquefies  blood  serum  and  forms  acid  in  dextrose, 
maltose  and  galactose  which  Mic.  catarrhalis  fails  to  do ; 
it  is  more  virulent  also  and  gives  rise  to  a  different  train  of 
symptoms.  The  difference  in  size,  however,  is  not  invariable, 
an  organism  no  greater  than  the  meningococcus  occasionally 
proving  on  examination  to  be  Mic.  catarrhalis  (Hachtel 
and  Hay  ward,  1911),  while  the  appearance  of  a  colony  is  a 
character  of  bacteria  liable,  as  we  have  shown,  to  undergo  great 
modification  (vide  p.  47). 

The  power  of  the  meningococcus  to  produce  fermentation 
in  sugars  is  subject  to  variation.  Arkwright  (1909)  found  that 


112  THE  POSSIBLE  OCCURRENCE  OF    [OH.  vm 

9  per  cent,  (out  of  36  cultures)  failed  to  produce  acid  in 
dextrose.  Summers  and  Wilson  (1909)  state  that  out  of 
80  strains  "nearly  all"  fermented  the  usual  sugars  but  a 
few  gave  the  fermentation  reactions  of  Mic.  catarrhalis.  The 
organism  isolated  from  sporadic  cases  of  meningococcal  menin- 
gitis shows  such  marked  differences  in  its  sugar  reactions 
when  compared  with  a  typical  meningococcus  that  some 
writers  regard  it  as  a  distinct  species  (Batten).  Arkwright 
(1909),  though  he  refutes  this,  acknowledges  that  the  sporadic 
type  is  less  uniform  in  its  fermenting  powers.  Some  of  his 
strains  of  meningococcus  permanently  failed  to  ferment  any 
sugars;  others,  which  failed  to  do  so  when  first  examined, 
gradually  acquired  the  power  in  the  course  of  many  months  ; 
others,  again,  which  did  ferment  sugars,  completely  lost  this 
property  after  cultivation  for  a  certain  time.  Another  interest- 
ing fact,  in  this  connection,  is  mentioned  by  Andrew  Connal 
(1910),  namely  that  in  the  late,  chronic  stages  of  cerebrospinal 
fever  the  meningococcus  isolated  from  the  cerebrospinal 
fluid  is  found  to  have  lost  its  power  to  break  up  sugar. 
Mic.  catarrhalis  on  the  other  hand  may  acquire  power  to 
ferment  sugars.  Gordon  (quoted  Martin,  1911)  found  that, 
out  of  25  strains  examined  by  him,  three  fermented  dextrose, 
saccharose,  galactose  and  maltose. 

The  meningococcus  and  Mic.  catarrhalis  differ  in  virulence 
but  this  property  in  the  latter  can  be  artificially  raised  by 
"passage." 

As  regards  pathogenesis,  this  distinction,  again,  between 
the  two  organisms  sometimes  breaks  down,  symptoms  typical 
of  infection  by  one  organism  being  in  reality  due  to  infection 
by  the  other.  The  symptoms  attributable  to  Mic.  catarrhalis 
infection  differ  widely.  Thus  it  may  cause  an  acute  pharyn- 
gitis (Gordon,  1906)  or  a  tonsilitis;  it  may  cause  a  "common 
cold"  or  give  rise  to  an  infective  cold  and  sore  throat  spreading 
from  person  to  person  (Allen,  1908);  it  may  set  up  otitis 
media  and  a  secondary  meningitis  (Barker,  1908),  or,  again,  a 
primary  meningitis  (Arkwright  and  Wilson)  or,  finally,  an 
epidemic  so  closely  resembling  cerebrospinal  fever  in  its 
symptoms  that  this  disease  has  actually  been  diagnosed  until 


CH.  vni]  TRANSMUTATION  IN  THE  LIVING  BODY  113 

a  bacteriological  examination  demonstrated  the  absence  of 
the  nieningococcus  and  the  presence  of  Mic.  catarrhalis 
(Dunn  and  Gordon,  1 905).  Conversely,  the  nieningococcus  may 
cause  a  simple  coryza  (20th  century  Diet,  of  Med.).  Sometimes 
a  "mixed  infection"  occurs;  thus,  Arkwright  (1909)  describes 
a  case  in  which  Mic.  catarrhalis  was  isolated  from  the 
heart's  blood  after  death  although  the  typical  nieningococcus 
was  proved  to  be  present  before  death  in  the  cerebrospinal 
fluid. 

Prof.  McDonald  (1908)  has  commented  upon  the  frequency 
with  which,  in  cerebrospinal  fever,  leptothrix  forms  are 
found  in  the  spinal  fluid  and  compares  this  with  the  similar 
frequency  of  leptothrix  forms  in  the  pharynx.  He  considers 
these  leptothrices  to  be  merely  secondary  invaders  but  regards 
their  presence  as  confirmatory  of  the  opinion,  now  generally 
held,  that  the  route  of  invasion  of  the  nieningococcus  in 
cases  of  cerebrospinal  fever  is  from  the  nasopharynx.  If  the 
appearance  of  these  "camp  followers"  tends  to  support  the 
opinion  as  to  the  locality  from  which  the  regiment  was 
drawn,  still  further  light  is  thrown  on  the  question  by  the 
presence  of  "disbanded  soldiers"  in  the  form  of  non- virulent 
meningococci  in  the  nasopharynx  of  healthy  persons.  Out  of 
a  total  of  810  healthy  persons,  examined  by  different  observers 
all  over  the  world,  the  nieningococcus  was  isolated  from  the 
nose  in  164  cases  (Hachtel  and  Hay  ward,  1911).  We  have 
already  referred  to  the  fact  that  organisms  normally  non- 
pathogenic  may  become  pathogenic  when  growing  and  multi- 
plying in  inflammatory  exudations  (vide  p.  79).  Cerebro- 
spinal fever  is  a  disease  more  particularly  of  young  children 
and  it  is  in  children  that  Mic.  catarrhalis  is  most  often 
discovered  as  an  inhabitant  of  the  pharynx  in  health.  It  has 
been  observed  that  an  attack  of  cerebrospinal  fever  very 
often  commences  with  a  purulent  nasal  discharge.  The  question 
arises,  does  this  area  of  suppurative  inflammation  in  the 
vicinity  of  its  natural  habitation  afford  a  training  ground,  so 
to  speak,  for  the  peaceful  Micrococcus  catarrhalis  preparatory 
to  its  entry  upon  a  military  career  in  the  uniform  of  the 
nieningococcus? 

D.  8 


114  THE  POSSIBLE  OCCURRENCE  OF    [CH.  vm 

The  oft  mooted  question  of  the  relationship  of  the  meningo- 
coccus to  the  pneumococcus  is  prompted  by  clinical  rather  than 
by  bacteriological  evidence.  Pneumococcal  meningitis,  like  all 
pneumococcal  infections,  is  characterised  by  certain  features 
which  are  also  observed  in  meningococcal  meningitis  (Preble), 
namely  an  acute  onset,  a  polymorphonuclear  leucocytosis, 
a  diminution  in  the  chlorides  in  the  urine,  and  herpes.  In  the 
second  place,  certain  complications  are  common  to  both,  namely 
endocarditis,  pericarditis,  arthritis  and  otitis  media.  In  the 
third  place,  Preble  observes  that  there  is  an  extraordinary 
similarity  in  the  seasonal  distribution  of  the  two  diseases. 
On  these  grounds  he  suggests  that  the  meningococcus  is  a 
variant  of  the  pneumococcus.  Certain  differences  between  the 
two  diseases  exist.  The  petechial  eruptions  which  formerly 
gave  a  name  to  one  disease  are  rare  in  the  other,  but  this  haem- 
orrhagic  tendency  is  altogether  absent  in  some  epidemics  of 
"meningococcal"  meningitis.  Again,  " meningococcal"  menin- 
gitis is  a  disease  more  especially  of  childhood  and  frequently 
ends  in  recovery;  "pneumococcal"  meningitis  is  a  disease 
more  commonly  of  adult  life  and  is  invariably  fatal.  The 
differences  in  age  incidence  and  mortality  are  however  com- 
patible with  the  view  that  the  causal  organism  is  the  same 
but  of  different  virulence. 

Finally,  the  sporadic  nature  of  meningococcal  meningitis, 
which  is  difficult  to'explain  if  one  admits  the  meningococcus 
to  be  an  independent  organism,  ceases  to  be  so  if  one  assumes 
it  to  be  a  modified  form  of  the  ubiquitous  pneumococcus. 

It  may  be  argued  that,  although  each  of  the  several  differ- 
ences in  character  which  distinguish  the  organisms  we  have 
been  comparing,  when  considered  by  itself,  may  appear*  trivial 
and  may  prove  to  be  variable,  nevertheless  all  these  differences, 
if  taken  together  and  viewed  as  a  whole,  represent  a  degree 
of  divergence  in  type  which  cannot  be  so  lightly  dismissed. 
A  series  of  surmises,  no  matter  how  credible  these  may  be 
made  to  appear,  does  not  constitute  a  proof.  It  is  in  our 
power  to  prove,  however,  that  in  other  cases  differences  no 
less  diverse  in  character  and  no  less  marked  in  degree,  differ- 
ences moreover  which,  taken  together  and  viewed  as  a  whole, 


CH.  vin]  TRANSMUTATION  IN  THE  LIVING  BODY  115 

might  be  thought  to  represent  no  less  wide  a  divergence  in 
type,  may  disappear  entirely  under  certain  conditions — con- 
ditions, be  it  noted,  precisely  analogous  to  those  which  we 
have  surmised  might  bring  about  a  similar  result  in  the  cases 
we  have  been  considering — namely,  invasion  of  the  living  body. 
The  experiments  of  Eyre,  Leatham  and  Washbourn  (1906) 
with  strains  of  the  pneumococcus  furnish  an  example.  These 
observers  describe  the  virulent,  parasitic  pneumococcus  as  re- 
quiring for  its  growth  a  certain  reaction  and  temperature,  and 
particular  media  (blood  agar) ;  it  would  not  grow  if  the  reaction 
were  even  faintly  acid  or  at  a  temperature  much  below  37°  C. 
and  rapidly  died  out  on  agar  or  in  broth.  It  would  not  liquefy 
gelatin  and  in  broth  formed  a  dust-like  deposit.  The  avirulent 
saprophytic  variety,  on  the  other  hand,  grew  luxuriantly  at 
temperatures  ranging  from  37°  to  20°  C. — on  agar,  gelatin, 
potato  or  in  broth,  whether  acid  or  alkaline,  slowly  liquefying 
gelatin  and  producing  a  uniform  turbidity  in  broth.  It  retained 
its  vitality  for  many  months.  It  also  exhibited  differences  in 
its  morphology, — "  instead  of  isolated  diplococci  and  strepto- 
cocci large  masses  of  cocci  and  diplococci  were  found  and 
forms  dividing  into  tetrads  were  common."  Nevertheless  this 
avirulent  saprophytic  pneumococcus  could,  by  a  single  "  pas- 
sage "  through  a  rabbit,  be  converted  into  a  typical  parasitic 
pneumococcus  of  high  virulence. 

Such  a  remarkable  transition,  if  it  did  not  actually  happen, 
would  seem  to  us  quite  as  improbable  as  a  transition  from, 
let  us  say,  the  micrococcus  catarrhalis  to  the  meningococcus. 

The  purpose  of  this  section  is  to  suggest  that  a  change  in 
character,  comparable  to  that  brought  about  in  the  case  of  the 
saprophytic  pneumococcus  by  a  single  animal  passage,  might 
be  brought  about  in  the  case  of  other  saprophytic  organisms 
by  an  analogous  process,  namely  by  their  invasion  of  the  living 
body  when  the  lowered  vitality  or  the  inflamed  condition  of 
the  tissues  enable  them  to  gain  a  foothold  therein. 


8—2 


CHAPTER  IX 

SUPPOSED  INSTANCES  OF  TEANSMUTATION 
BROUGHT  ABOUT  EXPERIMENTALLY 

I.    MAJOR  HORROCKS' s  EXPERIMENTS.  (Journal  of  R.A.M. C. 
Vol.  XVI.) 

In  March,  1911,  Major  Horrocks  published  the  records  of 
a  series  of  experiments  of  great  interest.  The  results  of  these 
experiments  may  be  briefly  summarised  as  follows  : 

(a)  From  a  strain  of  B.   typhosus  (derived  from  the 
urine  of  a  carrier)  he  obtained,  by  subculture,  an  organism 
intermediate  in    its  characters  between  B.   typhosus  and 
B.  coli. 

(b)  From  a  second  (laboratory)  strain  of  B.  typhosus,  by 
symbiosis  with  .R  coli,  he  obtained  an  organism  which  produced 
slight  acidity  in  mannite  but  fermented  no  other  sugars,  and 
which  later  reverted  to  J5.  typhosus. 

(c)  From  a  third  (laboratory)  strain  of  B.  typhosus,  by 
symbiosis  with  the  same  strain  of  B.  coli,  he  obtained  an  or- 
ganism closely  resembling  B./aecalis  alcaligenes. 

(d)  From  a  fourth  strain  of  B.  typhosus  (derived*  from  the 
urine  of  another  carrier),  after  injection  into  the  peritoneal 
cavity  of  a  guineapig,  he  obtained  a  Gram-positive  coccus 
resembling  streptococcus  faecalis. 

(e)  From  a  fifth  strain  of  B.  typhosus  (derived  from  the 
urine  of  a  third  carrier),  after  injection  into  the  peritoneal 
cavity  of  a  guineapig,   he  obtained  in  three  different  ex- 
periments a  coliform  organism  which  differed  widely  in  its 
fermentation  and  agglutination  properties  from  B.  typhosus. 

(/)  From  a  sixth  strain  of  B.  typhosus  (derived  from  the 
stool  of  a  fourth  carrier),  after  growth  in  the  diluted  and 
filtered  urine  of  another  carrier  "S,"  he  obtained  B./aecalis 


OH.  ix]          SUPPOSED  TRANSMUTATIONS  117 

alcaligenes.  The  latter  organism  in  one  experiment,  after 
three  passages  through  the  guineapig,  gave  rise  to  B.  coli 
which  however  reverted  subsequently  to  B.  faecalis  alcali- 
genes. 

(g)  From  the  second  (laboratory)  strain  of  B.  typhosus 
referred  to  above  (b\  after  exposure  to  the  same  conditions, 
he  obtained  in  two  different  experiments  B.  faecalis  alcali- 
genes. The  latter  organism  in  two  later  experiments  (in  the 
first  after  5  months'  further  growth  and  in  the  second  after 
8  passages  through  the  guinea-pig)  gave  rise  to  streptococcus 
faecalis ;  while  in  a  third  experiment  (after  18  successive 
passages  through  the  guinea-pig)  it  gave  rise  to  B.  coli  which, 
after  the  19th  passage,  reverted  to  B.  faecalis  alcaligenes  and 
this,  after  further  "passages,"  in  two  different  experiments 
yielded  the  streptococcus  faecalis. 

(h)  From  the  same  (laboratory)  strain  of  B.  typhosus,  after 
growth  in  the  diluted  and  filtered  urine  a  different  carrier 
"  I,"  he  obtained  again  B.  faecalis  alcaligenes. 

To  summarise  these  results  even  more  concisely,  it  appears 
that  Major  Horrocks  was  forced  to  the  conclusion  that  not 
only  had  an  organism  arisen  from  a  strain  of  B.  typhosus  in- 
termediate in  character  between  B.  typhosus  and  B.  coli,  but 
that  other  strains  of  B.  typhosus,  derived  from  three  distinct 
sources,  had  in  no  less  than  five  of  his  experiments  undergone 
mutation  into  B.  faecalis  alcaligenes  as  a  result  of  changes 
in  their  environment ;  and,  further,  that  the  B.  faecalis  alcali- 
genes so  obtained  had  later,  in  two  instances,  become  changed 
into  B.  coli  (reversion  taking  place  in  both  cases,  however, 
subsequently)  and,  in  four  instances,  become  changed  into 
streptococcus  faecalis,  once  after  prolonged  cultivation  and 
three  times  as  the  result  of  passage ;  and,  finally,  that  one 
of  the  original  strains  of  B.  typhosus  had  undergone  a  similar 
change  into  streptococcus  faecalis  after  passage. 

Major  Horrocks's  statements  are  so  startling  and,  if  sub- 
stantiated, would  prove  so  revolutionary  in  character  that  they 
demand  careful  examination. 

It  may  not  be  possible  to  disprove  either  his  facts  or  his 
inferences,  but  it  is  not  necessary  to  do  so.  The  onus  of  proof 


118  SUPPOSED  INSTANCES  [CH.  ix 

rests  with  the  claimant.  If  it  is  possible  to  show  that  he  has 
failed  to  exclude  a  single  possible  source  of  error,  a  verdict  of 
not  proven  must  be  returned. 

When  considering  the  value  of  evidence  adduced  in  sup- 
port of  supposed  instances  of  variation  or  transmutation  (vide 
Chap.  Ill)  we  mentioned  various  sources  of  error.  Bearing  these 
in  mind,  and  also  the  wide  limits  within  which  we  have  found 
variation  may  occur  (vide  Chaps.  IY-VII)  we  will  now  consider 
in  detail  the  processes  by  which  Major  Horrocks  obtained  the 
results  he  claimed  and  the  value  of  the  evidence  he  brings 
forward  to  support  his  contentions. 

(a)  The  alteration  of  B.  typhosus  to  an  organism  inter- 
mediate between  B.  typhosus  and  B.  coli. 

(Page  246.)  A  strain  of  B.  typhosus  was  isolated  from 
the  urine  of  a  typhoid  carrier  "  TS  " — from  whose  blood  a  pure 
culture  of  B.  typhosus  had  previously  been  obtained.  After 
3  days'  incubation  on  bile  salt  glucose  litmus  agar  the  strain 
gave  the  typical  reactions  of  B.  typhosus.  At  the  end  of  a 
week,  however,  the  following  characters  were  displayed :  lac- 
tose, salicin  and  dulcite  were  rendered  slightly  acid,  broth 
gave  a  marked  indol  reaction,  the  neutral  red  reaction  yielded 
a  slight  yellow  colouration,  the  organism  appeared  only  slightly 
motile  and  was  not  agglutinated  by  anti-typhoid  serum.  The 
organism,  however,  gave  rise  to  typhoid  agglutinins  when 
injected  into  a  rabbit,  and  this  rabbit's  serum  deviated  com- 
plement in  the  same  manner  as  a  known  antityphoid  serum, 
and  the  organism  further  had  the  power  of  absorbing  the 
specific  agglutinins  from  a  known  typhoid  serum. 

After  4  passages  through  the  guineapig  the  organism  lost 
its  lactose  fermenting  property  and  only  differed  from  the 
original  B.  typhosus  by  forming  a  trace  of  acid  in  salicin. 
After  4  further  passages  it  reverted  to  the  unusual  fermenting 
type  described. 

The  urine  of  carrier  "  TS  "  from  which  the  strain  was  origin- 
ally derived  was  again  carefully  tested  but  only  typical  typhoid 
organisms  were  obtained. 

Criticism.  We  have  already  quoted  (vide  p.  11)  instances 
of  the  occurrence  of  organisms,  derived  in  some  cases  from 


CH.  ix]  OF  TRANSMUTATION  119 

the  urine  of  typhoid  carriers,  intermediate  in  character  between 
B.  typhosus  and  B.  coli.  Wilson  (1910)  described  such  an 
organism  as  fermenting  glucose  and  mannite  but,  unlike  B.  ty- 
phosus, fermenting  lactose  also  at  22°  C.  (but  not  at  37°  C.) 
and  failing  to  agglutinate  with  typhoid  serum.  The  Bacillus 
perturbans  of  Klotz  (1906),  though  agglutinated  by  high  dilu- 
tions of  typhoid  serum,  fermented  lactose  and  saccharose, 
gave  the  neutral  red  reaction  and  produced  indol. 

Examples  have  also  been  given  (vide  Chapter  V)  to  show 
the  variability  of  organisms  with  respect  to  their  power  to 
ferment  sugars  and  their  ability  to  acquire  fresh  fermenting 
properties.  B.  typhosus,  for  example,  may  acquire  the  power 
in  a  few  days  to  ferment  dulcite. 

The  organism  described  here  by  Major  Horrocks  is  another 
example  of  temporary  variation  in  character  with  respect  to  the 
power  to  ferment  sugars  and  to  produce  indol,  associated  with 
some  modification  also  in  agglutination  properties. 

(&,  c)  The  change  from  B.  typhosus  to  B.  faecalis  alcaligenes 
due  to  symbiosis  with  B.  coli. 

(Page  233,  exp.  1.)  The  strain  of  B.  typhosus  used  was 
a  stock  laboratory  strain  "  R,"  from  which  stock  vaccines  were 
prepared — a  strain,  that  is  to  say,  of  unimpeachable  character. 
The  strain  of  B.  coli  was  derived  from  the  urine  of  a  typhoid 
carrier  ("Bomb  S  ").  The  two  organisms  were  added  to  1  c.c.  of 
sterilised  tap  water  and  the  suspension  plated.  10  days  later, 
examination  showed  typical  typhoid  colonies  and  others  white 
and  opaque.  The  latter  were  planted  on  the  usual  media  and 
in  48  hours  yielded  slight  acidity  in  mannite  only ;  no  other 
sugars  were  fermented  in  7  days.  The  original  strain  of  B. 
typhosus  used  was  replanted  on  agar  and  the  resulting  growth 
gave  the  typical  reactions  of  this  organism. 

(Page  234,  exp.  3.)  The  experiment  was  repeated,  a  dif- 
ferent typhoid  strain  ("Bombay  ")  being  used.  After  an  interval 
of  two  months,  1  c.c.  of  the  inoculated  water  was  added  to 
MacConkey's  bile  salt  broth  and  this  plated  on  lactose  bile 
salt  litmus  agar.  A  few  blue  colonies  were  seen  consisting  of 
bacilli  which  resembled  B.  faecalis  alcaligenes  in  not  ferment- 
ing any  sugars  and  producing  an  alkaline  reaction  in  milk. 


120  SUPPOSED  INSTANCES  [OH.  ix 

No  B.  typhosus  could  be  isolated  and  at  later  examinations 
only  B.  coli  was  recovered. 

Criticism.  Conditions  of  growth  inimical  to  the  life  of  an 
organism  might  be  expected  to  deprive  it  gradually  of  its 
functions.  A  strain  of  typhoid  bacilli  whose  vitality  is  at  its 
lowest  ebb  would  hardly  be  likely  to  ferment  sugars  vigorously, 
if  at  all.  In  both  these  experiments  two  factors  were  at  work 
inimical  to  the  life  of  B.  typhosus,  namely  the  presence  of 
B.  coli,  and  growth  in  water — a  non-nutrient  medium.  After 
10  days,  in  the  first  experiment,  slight  fermenting  power 
persisted.  After  two  months,  in  the  second  experiment,  all 
fermenting  power  was  lost.  No  attempt  was  made  to  resusci- 
tate the  strain  of  organisms  on  ordinary  media  to  ascertain 
whether  with  returning  vitality  fermenting  power  would  be 
restored. 

That  this  explanation  is  the  true  one  and  that  no  new  race 
of  organisms  was  produced  is  suggested  further  by  the  obser- 
vation that  no  organisms  giving  the  ordinary  reactions  of  B. 
typhosus  survived  side  by  side  with  the  non-fermenters  and 
that,  at  a  later  stage,  the  strain  was  found  to  have  died  out 
altogether. 

(d)  The  change  from  B.  typhosus  to  Streptococcus  faecalis 
in  the  peritoneal  cavity  of  the  guineapig. 

(Page  230,  exp.  4.)  The  urine  of  a  typhoid  carrier  "S" 
was  plated  and  found  to  contain  typhoid  bacilli.  One  colony 
was  subcultured  on  agar  and  a  standard  loopful  of  a  24  hours' 
growth  was  injected  into  the  peritoneal  cavity  of  a  guineapig. 
The  animal  was  found  dead  in  the  morning.  No  typhoid  ba- 
cilli were  found  in  the  peritoneal  fluid  which  contained  a  pure 
culture  of  a  Gram-positive  streptococcus  giving  the  reactions 
of  S.  faecalis. 

(e)  The  change  in  the  peritoneal  cavity  of  a  guineapig 
from  B.  typhosus  to  a  coliform  organism  giving  atypical  re- 
actions. 

The  urine  of  a  typhoid  carrier  "I"  was  plated  after  being 
kept  12  months  in  a  flask.  Two  colonies  of  B.  typhosus  were 
planted  on  agar  and  labelled  IBl  and  IB2  respectively. 

(e  i)  (Page  230,  exp.  6.)  One  standard  loopful  of  a  24  hours' 


CH.  ix]  OF  TRANSMUTATION  121 

growth  from  the  culture  IBl  was  injected  into  the  peritoneal 
cavity  of  a  guineapig.  The  animal  was  found  dead  the  next 
morning  and  a  pure  culture  of  B.  typhosus  was  obtained  from 
the  heart's  blood.  From  the  peritoneal  fluid  and  spleen  was 
obtained,  in  addition  to  B.  typhosus,  a  coliform  organism 
possessing  the  following  characters :  a  gram-negative  motile 
bacillus,  forming  acid  and  gas  in  glucose,  mannite,  lactose  and 
dulcite,  but  producing  no  change  in  salicin  or  cane  sugar  but 
giving  rise  in  the  neutral  red  medium  to  gas  and  fluorescence, 
not  liquefying  gelatin  or  forming  indol  in  broth  and  giving  an 
acid  reaction  in  litmus  milk  without  any  clotting.  A  broth 
culture  from  the  original  agar  slope  was  carefully  tested  but 
typical  B.  typhosus  alone  found.  The  broth  culture  was 
planted  on  agar  and  the  experiment  repeated  with  a  loopful 
of  this  growth.  The  animal  did  not  die  and  a  pure  culture 
of  B.  typhosus  was  recovered  from  the  peritoneal  cavity. 

(e  ii)  (Page  230,  exp.  6.)  One  standard  loopful  of  a  24  hours' 
growth  from  the  culture  IB2  was  then  injected  into  the  peri- 
toneal cavity  of  a  guineapig  in  the  same  manner.  The  animal 
was  found  dead  next  morning  and  a  pure  culture  of  B.  typhosus 
was  obtained  from  the  heart's  blood  and  spleen.  From  the 
peritoneal  fluid  was  obtained,  in  addition  to  B.  typhosus,  a 
coliform  bacillus. 

(e  iii)  (Page  231,  exp.  7.)  The  urine  of  the  typhoid  carrier 
"  I "  was  again  plated  after  having  been  kept  over  14  months 
in  a  flask.  A  colony  was  again  planted  on  agar  and  a  standard 
loopful  of  the  growth  again  injected  into  the  peritoneal  fluid 
of  a  guineapig.  The  animal  was  found  to  be  dying  the  next 
morning  and  was  killed  with  chloroform  and  a  pure  culture 
of  B.  typhosus  was  obtained  from  the  heart's  blood.  From  the 
peritoneal  fluid  and  spleen  a  pure  culture  of  a  coliform  or- 
ganism was  obtained.  The  latter  organism  failed  to  produce 
any  typhoid  agglutinins  when  injected  into  a  rabbit,  or  to  ab- 
sorb agglutinins  from  a  known  typhoid  serum. 

The  last  experiment  was  repeated,  the  same  strain  being 
used  ("  I ")  after  14  days  further  growth  on  agar.  The  injection 
did  not  prove  fatal  to  the  guineapig  and  from  the  peritoneal 
fluid  a  pure  culture  of  B.  typhosus  was  obtained. 


122  SUPPOSED  INSTANCES  [CH.  ix 

Criticism.  The  last  four  experiments  (d,  e  i,  e  ii,  and  e  iii) 
may  be  discussed  together. 

In  one  experiment  (d)  B.  typhosus  derived  from  a  carrier 
apparently  gave  rise,  in  the  peritoneal  cavity,  to  a  Gram-positive 
coccus.  In  three  experiments  (e  i,  e  ii,  and  e  iii)  B.  typhosus, 
derived  from  another  carrier,  apparently  gave  rise,  in  the  peri- 
toneal cavity,  to  atypical  coliform  organisms. 

The  questions  to  be  discussed  are  two — whether  the  strain 
of  organisms  isolated  from  the  peritoneal  cavity  were  derived 
from  the  original  strain  of  B.  typhosus  injected  in  each  case, 
and  whether,  if  such  continuity  is  established,  the  alteration 
in  character  is  to  be  regarded  as  a  temporary  variation  or  a 
transmutation. 

The  possibilities  to  be  considered  are  (1)  whether  the 
original  strain  of  B.  typhosus  was  pure ;  (2)  whether  the  peri- 
toneal cavity  in  each  case  was  sterile  before  the  injection  was 
made ;  (3)  whether  it  was  contaminated  from  the  skin  at  the 
time  the  injection  was  made ;  (4)  whether  it  was  invaded  from 
the  gut  after  the  injection  was  made  or  after  the  death  of  the 
animal ;  (5)  whether  the  later  strain  was  linked  up  with  the 
original  one  by  the  occurrence  of  reversion  or  the  discovery 
of  intermediate  forms;  (6)  whether  a  repetition  of  the  ex- 
periments confirmed  the  results;  (7)  whether  the  alteration 
in  character  falls  within  the  recognised  limits  of  variation 
discussed  in  the  earlier  part  of  this  work. 

(1)  There  are  grounds  for  viewing  the  original  cultures  with 
suspicion.  They  were  not  made  in  any  instance  from  a  single 
organism.  The  urine  of  carrier  "S"  and  of  carrier  "I,"  from 
which  they  were  isolated,  admittedly  contained  streptococci, 
B.  coli,  bacilli  closely  resembling  B.  faecalis  alcaligenes  and 
other  coliform  organisms.  These  other  organisms  were  present 
in  comparatively  small  numbers.  In  one  instance  it  is  stated 
that  B.  coli  and  B.  typhosus  were  present  in  the  proportion 
of  1  to  30,000.  Such  disparity  in  numbers  might  easily  account 
for  the  less  common  organisms  being  overlooked.  It  is  men- 
tioned that  a  change  in  the  character  of  the  medium,  brought 
about  by  simple  dilution  with  water,  enabled  the  associated 
microbes  to  multiply  so  much  more  rapidly  than  the  B.  ly- 


CH.  ix]  OF  TRANSMUTATION  123 

phosus  that  the  latter  organism  was  soon  "swamped,"  as  it 
were,  and  disappeared  altogether.  If  the  strain  of  B.  typhosus 
injected  contained  one  or  two  specimens  of  a  streptococcus  or 
coliform  organism,  might  not  growth  in  the  peritoneal  cavity 
yield  a  similar  result? — not  before,  however,  some  of  the  ty- 
phoid bacilli  had  succeeded  in  escaping  from  the  peritoneum 
into  the  blood  vessels  and  setting  up  a  systemic  infection.  In 
two  instances,  in  which  the  experiments  were  repeated,  the 
injection  of  the  original  culture  of  B.  typhosus  into  the  peri- 
toneal cavity  did  not  kill  the  animal  although  a  pure  culture 
of  B.  typhosus  was  recovered  from  it.  The  original  culture, 
therefore,  apparently  contained  strains  differing  from  each 
other  in  virulence.  They  may  conceivably  have  possessed 
other  differences. 

(2)  No  control  experiments  were  carried  out  to  prove  that 
the  peritoneal  cavity  before  the    experiment  was    sterile. 
Dudgeon  (1908)  states  that  in  healthy  animals  the  omentum 
may  normally  contain  the  staphylococcus  albus.    There  is 
evidence  to  show  that  even  in  healthy  animals  the  internal 
organs  may  contain   both   pathogenic  and  non-pathogenic 
bacteria.  Ford  (1900)  showed  by  experiments,  in  which  rigid 
precautions  against  contamination  were  adopted,  that  the 
kidneys,  liver  and  spleen  of  healthy  animals,  in  a  large  majority 
of  cases,  contained  organisms  such  as  the  staphylococcus, 
mesentericus,  colon  and  paracolon   bacilli,  B.  subtilis  and 
proteus.    In  rabbits  66  per  cent,  of  the  organs  examined 
contained  bacteria,  in  cats  over  77  per  cent,  in  dogs  over  88 
per  cent.   In  guineapigs  the  percentage  was  77  per  cent,  of 
the  organs  examined  and  the  organisms  that  predominated 
were  B.  subtilis(  staphylococci  and  the  colon  bacillus.  Adami, 
Abbott  and  Nicholson  (1899)  found  in  the  livers  of  healthy 
animals  (cows,   sheep,   rabbits,   guineapigs)   diplococci  and 
chains  of  3  or  4  cocci,  which  on  culture  yielded  B.  coli  in 
many  cases. 

(3)  No  control  experiments  were  conducted  to  exclude 
the  possibility  of  skin  contamination  at  the  site  of  the 
inoculation,  but  such  a  supposition  is  inadequate  to  explain 
all  the  results  obtained. 


124  SUPPOSED  INSTANCES  [CH.  ix 

(4)  The  injection  of  organisms  into  the  peritoneal  cavity 
would  have  a  threefold  effect,  it  would  make  the  animal  ill, 
produce  a  more  or  less  marked  peritonitis,  and  finally  kill 
the  animal.  All  three  events  would  favour  the  invasion  of 
the  peritoneal  cavity  by  organisms.  If  the  vitality  of  the 
body  and  consequently  of  the  peritoneum  is  lowered,  organisms 
can  penetrate  it  from  the  gut  even  in  the  absence  of  any 
definite  lesion  or  inflammation.  Ford  (1900)  noted  that  in 
animals  whose  vitality  was  lowered,  by  fasting  or  unhealthy 
conditions,  bacteria  were  more  abundant  in  the  internal 
organs,  and  that  this  applied  particularly  to  bacteria  of  the 
colon  type.  Dudgeon  and  Sargent  (1907)  have  shown  that  at 
the  earliest  stage  of  peritonitis  the  staphylococcus  albus 
(either  normally  present  on  the  surface  of  the  gut  or  pene- 
trating from  within  it)  increases  with  enormous  rapidity. 
Miiller  (1910)  remarks  that  when  organisms  (e.g.  typhoid 
bacilli)  are  injected  into  the  peritoneal  cavity  they  at  first 
decrease  in  number  owing  to  the  bactericidal  effect  of  the 
body  fluids  but  later  on  increase  again.  It  is  during  this 
early  stage  when  the  injected  organisms  are  decreasing  rapidly 
that  the  staphylococcus  albus  (and  possibly,  in  animals,  the 
bacteria  present  in  the  internal  organs)  are  increasing  rapidly. 
A  culture  removed  during  this  period  might  well  convey  the 
impression  that  a  mutation  had  occurred. 

Dudgeon  and  Sargent  (1907)  mention  that  the  staphylo- 
coccus albus  is  often  quite  non-pathogenic  in  the  peritoneal 
cavity  of  the  guineapig. 

After  death  there  is  a  rapid  invasion  of  the  peritoneal 
cavity  by  organisms,  particularly  by  B.  coli  from  the  gut,  so 
that  the  true  nature  of  the  infection  becomes  obscured.  In 
illustration  of  this  point,  Dudgeon  and  Sargent  (1907)  record 
a  case  of  pneumococcal  peritonitis  in  which  the  peritoneal 
exudate  one  hour  after  death  gave  a  pure  culture  of  pneumo- 
cocci,  whereas  26  hours  later  B.  coli  alone  could  be  recognised 
in  the  same  exudate. 

It  is  not  possible,  therefore,  to  exclude  in  Major  Horrocks's 
experiments  the  possibility  of  an  invasion  of  the  peritoneal 
cavity  from  the  gut,  following  either  the  inoculation  or  the 
death  of  the  animal. 


CH.  ix]  OF  TRANSMUTATION  125 

(5)  In  none  of  these  four  experiments  was  any  attempt 
made  to  test  the  new  strain  by  subculture  or  passage  to 
ascertain  whether  it  would  revert.   In  two  experiments  the 
original  strain  of  B.  typhosus  had,  within  a  few  hours  of  its 
injection,  completely  disappeared  and  in  none  of  the  experi- 
ments were  any  intermediate  forms  observed  which  might  be 
regarded  as  linking  up  the  new  strain  with  the  original  one 
and  suggesting  a  transmutation.     All  the  organisms  were 
apparently  of  the  same  type.    Moreover  the  agglutination 
reactions  betrayed  no  sign,  in  the  only  instance  in  which  they 
were  tested,  of  any  connection  between  the  new  strain  and 
the  original  B.  typhosus.    The  continuity,  therefore,  of  the 
two  forms  cannot  be  regarded  as  proved.    Moreover  if  the 
variants  were  really  derived  from  the  original   strain    of 
B.  typhosus  one  would  have  expected  them  to  be  present,  like 
the  typhoid  bacilli,  in  the  blood  stream  as  well  as  in  the 
peritoneal  cavity.  One  possible  explanation  is  that  the  change 
was  dependent  upon  the  influence  of  some  agent  existing  in 
the  peritoneum  but  absent  elsewhere.  This  will  be  referred 
to  again  (vide  p.  126). 

(6)  A  repetition  of  the  experiments  was  made  in  only  two 
cases  (e\  and  ciii)  and  without  yielding  similar  results — 
indeed  the  results  differed  from  those  first  obtained  in  such  a 
way  as  to  suggest  that  the  original  cultures,  in  both  cases, 
contained  strains  of  bacteria  differing  widely,  at  any  rate  in 
their  virulence. 

(7)  If  the  continuity  of  the  two  different  strains  were 
established  in  each  case,  what  would  be  the  significance  of 
these  changes  ?  The  transition  from  B.  typhosus  to  an  atypical 
coliform  organism  may  be  regarded  as  a  variation  on  the  part 
of  the  typhoid  strain,  probably  temporary  in  character  and 
of  the  same  nature  as  those  discussed  in  an  earlier  part  of 
this  work  (vide  p.  11).  The  transition  from  B.  typhosus  to  a 
Gram-positive  coccus  is  more  difficult  to  explain  but  the 
observations  of  Adami  and  others  would  suggest  that,  in  this 
case  also,  a  temporary  variation  and  not  a  true  transmutation 
might  have  been  brought  about.  Adami  (1892)  observed  that 
the  addition  to  a  medium  of  substances  inimical  to  the  life 


126  SUPPOSED  INSTANCES  [CH.  ix 

of  B.  typhosm — for  example,  carbolic  acid  or  creosote — 
made  this  bacillus  in  such  a  medium  assume  temporarily  the 
form  of  non-motile  cocci  or  diplococci.  Again,  Adami,  Abbott 
and  Nicholson  (1899)  obtained  from  the  bile  in  guineapigs 
and  also  from  the  peritoneal  fluid  in  man,  under  certain 
conditions,  coccic  forms  of  B.  coli.  These  were  present  as 
diplococci  or  short  chains  of  3  or  4  cocci ;  they  were  non-motile, 
non-fermenting  and  did  not  produce  indol ;  their  growth  on  the 
surface  of  agar  at  first  closely  resembled  that  of  a  strepto- 
coccus, the  colonies  were  white  and  opaque.  Intraperitoneal 
inoculation  into  a  guineapig  increased  their  fermenting  power 
and,  after  3  passages,  yielded  normal  B.  coli.  They  found 
evidence  that  B.  typhosm  yielded  similar  modified  coccic 
forms  when  acted  on  by  peritoneal  and  other  fluids.  They 
describe  coccic  forms  of  B.  typhosm  in  the  mesenteric  and 
retroperitoneal  glands.  They  mention  that  in  some  cases  the 
action  of  ascitic  and  peritoneal  fluids  in  this  respect  is  so 
marked  that  it  was  difficult  to  obtain  complete  reversion  to 
type.  They  found,  also,  that  B.  coli  injected  into  the  blood 
stream  in  a  rabbit  appeared  in  these  coccic  forms  within  half 
an  hour  in  the  liver  and  the  bile,  though  similar  forms  were 
not  found  in  the  systemic  circulation.  If  the  modification  in 
a  strain  of  B.  typhosm  which  Major  Horrocks  describes  was 
due  to  the  same  agency,  one  can  understand  why  the  variant  was 
only  found  in  the  peritoneal  cavity  and  not  in  the  heart's 
blood.  In  his  later  experiments,  however,  cocci  possessing  the 
characters  of  S.  faecalis  were  obtained  not  only  in  the 
peritoneal  cavity  but  during  culture  on  artificial  media.  In 
the  account  of  these  experiments,  however,  there  is  much  to 
suggest  that  the  original  strain  was  not  a  pure  one. 

(f)  The  change  from  B.  typhosus  to  B.  faecalis  alcaligenes 
after  growth  in  the  diluted  and  filtered  urine  of  a  typhoid 
carrier  and  the  further  changes  from  B.  faecalis  alcaligenes 
to  B.  coli  on  passage. 

(Page  237,  exp.  I.)  The  urine  of  a  typhoid  carrier  "  S  " 
was  diluted  1  in  10  with  tap  water  and  allowed  to  stand  11 
days.  It  was  then  filtered  through  a  Pasteur  candle  (F)  with- 
out pressure  and  shown  to  be  sterile  by  "prolonged  incubation" 


CH.  ix]  OF  TRANSMUTATION  127 

at  37°  C.  after  plating.  The  filtrate  was  then  inoculated  with 
a  48  hours'  growth  of  B.  typhosus  isolated  from  the  stool  of  a 
carrier  "  C  "  and  a  week  later  plated  out  on  bile  salt  neutral 
red  lactose  agar.  Two  or  three  colonies  were  examined.  One 
gave  the  typical  reactions  of  B.  typhosus  but  two  other 
colonies  failed  to  agglutinate  with  typhoid  serum  and  cor- 
responded in  their  reactions  to  B.  faecalis  alcaligenes.  One 
week  later  the  latter  organism  alone  was  found  and  it 
persisted  unchanged  for  several  months  afterwards. 

Criticism.  The  same  criticism  applies  to  this  experiment. 
The  original  strain  was  not  grown  from  a  single  organism. 
"A  particle,"  we  are  told,  of  the  growth  was  added  to  the 
solution,  sufficient  to  yield  480  million  bacteria  to  each  cubic 
centimetre.  The  purity  of  such  a  strain  cannot  be  guaranteed. 
The  original  culture  was  derived  from  a  stool  and  it  is  said 
to  have  yielded  an  organism  closely  resembling  B.  faecalis 
alcaligenes.  The  experiment  was  however  repeated  twice 
over  with  a  laboratory  strain  with  the  same  result — a  fact 
which  considerably  discounts  this  objection.  The  new  strain 
might,  again,  be  regarded  as  a  variant  of  the  original  B. 
typhosus  which,  as  a  result  of  growth  under  conditions 
inimical  to  its  vitality,  had  suffered  a  loss  of  power  with 
respect  to  its  fermenting  properties  and  also  its  property  of 
agglutinating  with  typhoid  serum.  Its  other  agglutinative 
characters  were  not  examined,  but  a  similar  organism  obtained 
in  the  same  manner  from  a  laboratory  strain  was  found  to 
possess  slight  power  of  absorbing  agglutinins  from  antityphoid 
serum.  The  new  strain  was,  however,  further  tested  ("  culture 
33  "  page  242)  by  being  alternately  passed  through  the  peri- 
toneal cavity  of  a  guineapig  and  subcultured  on  agar.  In  one 
experiment,  after  the  3rd  passage,  the  peritoneal  fluid  removed 
at  the  end  of  6  hours  contained  B.  coli,  but  the  fluid  removed 
at  the  end  of  12  hours  contained  not  B.  coli  but  the  new 
strain  of  B.  faecalis  alcaligenes  which  had  been  injected. 

Only  three  passages  were  made — the  investigation,  that  is 
to  say,  was  not  persisted  in  long  enough  to  decide  whether 
the  new  strain  was  capable  of  reverting  or  not. 

The  recovery  of  B.   coli  after  the  3rd  passage  Major 


128  SUPPOSED  INSTANCES  [CH.  ix 

Horrocks  considers  may  have  been  due  to  an  invasion  by  this 
organism  from  the  gut  but  was  more  probably  due,  in  his 
opinion,  to  the  still  further  modification  of  the  strain  injected, 
inasmuch  as  "  reversion  "  apparently  took  place  a  few  hours 
later.  The  disappearance  of  the  B.  coli  might  however  be 
explained  on  other  grounds.  The  strain  of  B.  faecalis 
alcaligenes  undergoing  passage  may  have  been  contaminated 
with  B.  coli  between  the  second  and  third  passages ;  the 
former  organism  after  its  two  passages  might  be  expected  to 
be  more  resistant  to  the  body  fluids  which  destroyed  the 
latter. 

(g)  The  change  from  B.  typhosus  to  B.  faecalis  alcaligenes 
after  growth  in  the  diluted  and  filtered  urine  of  a  typhoid 
carrier  and  the  further  change  from  B.  faecalis  alcaligenes 
to  Streptococcus  faecalis. 

(Page  238,  exp.  II.)  The  sterile  filtrate  from  the  urine 
of  a  typhoid  carrier  "S"  was  again  inoculated  with  B. 
typhosus — the  strain  used,  on  this  occasion,  being  a  stock 
laboratory  strain  "R"  of  unimpeachable  character.  The 
result  was  similar  to  that  in  the  previous  experiment.  Side 
by  side  with  colonies  of  typical  B.  typhosus  were  found 
colonies  of  an  organism  ("  35  A.  Col.  2  ")  which  corresponded 
with  B.  faecalis  alcaligenes  but  possessed  a  slight  power  of 
absorbing  agglutinins  from  antityphoid  serum  (p.  242).  Later 
the  latter  organism  alone  was  found  and  it  persisted  unchanged 
for  many  months.  The  experiment  was  repeated  in  exactly 
the  same  way  and  with  the  same  result,  B.  faecalis  al- 
caligenes emerging  (p.  240,  exp.  IV). 

These  two  experiments  are  simply  a  repetition  of  the 
experiments  already  discussed,  a  laboratory  strain  of  B. 
typhosus  being  used  instead  of  a  carrier  strain. 

When  the  inoculated  filtrate  used  in  the  first  experiment 
(p.  238,  exp.  II)  was  6  months  old,  a  loopful  of  it  was  added 
to  a  broth  tube  and  a  48  hours'  growth  plated  on  MacConkey's 
medium  (p.  239).  Colonies  of  B.  faecalis  alcaligenes  were 
again  found  but  with  them  smaller  colonies  of  a  streptococcus 
closely  resembling  8.  faecalis. 

The  strain  of  B.  faecalis  alcaligenes  obtained  in  the 


CH.  ix]  OF  TRANSMUTATION  129 

first  experiment  (p.  238,  exp.  II)— known  after  this  as  "35  A  "- 
was  further  tested  (p.  243)  by  successive  passages  through 
the  peritoneal  cavity  of  the  guineapig.  The  fluid  removed 
after  the  8th  passage  when  subcultured  into  broth  showed 
short-chained  cocci  and  diplococci  but  when  subcultured  on 
to  agar  gave,  in  addition  to  these  cocci,  the  original  bacillus 
"  35A."  The  latter  in  broth  again  yielded  the  short-chained 
cocci  and  these  on  agar  gave  a  bacillus  once  more — but  this 
time  not  "  35  A  "  but  a  fermenting  coliform  organism. 

The  original  strain  of  B.  faecalis  alcaligenes  (obtained 
in  Exp.  II,  p.  238)  was  a  second  time  tested  in  the  same  way 
(p.  243).  It  showed  no  change  in  character  until  the  18th 
passage  when  it  gave  rise  to  a  fermenting  bacillus  of  the  B. 
coli  type  which  on  the  19th  passage  reverted  to  B.  faecalis 
alcaligenes.  This  last-named  organism,  after  7  more  passages 
in  one  experiment  and  8  more  passages  in  another,  gave  rise 
to  a  fermenting  B.  coli  type  of  organism  "  in  pure  culture  " 
and  this,  on  planting  in  broth,  yielded  B.  faecalis  alcaligenes 
once  more  but,  with  it,  cocci  corresponding  to  S.  faecalis. 
The  latter  after  3  passages  remained  unchanged. 

Criticism.  The  transition  from  the  non-fermenting  B. 
faecalis  alcaligenes  to  the  fermenting  coli  type  and  back 
again,  may  be  regarded  as  no  more  than  another  example  of 
variation  similar  to  those  quoted  in  an  earlier  section  of  this 
work.  Again,  in  the  "  passage  "  experiments,  one  or  other 
type  may  have  been  an  invader  from  the  gut,  the  apparent 
reversion  at  a  later  passage  merely  representing  the  de- 
struction of  the  invader  which  had  not  been  "  hardened/'  so 
to  speak,  by  previous  passages.  Only  one  guineapig  was  used 
for  each  "  passage "  experiment  in  a  series  of  passages.  If 
more  than  one  had  been  used  some  check  would  have  existed 
on  possible  errors  due  to  this  cause.  The  repeated  transitions 
from  B.  faecalis  alcaligenes  to  &  faecalis  and  vice  versa 
are  more  difficult  to  explain,  for  8.  faecalis  appeared 
not  only  after  passage  but  during  cultivation  also  on  artificial 
media.  One  is  almost  forced  to  the  conclusion  that  Major 
Horrocks  was  dealing  with  a  mixed  stram  of  the  two  organisms 
and  that  changes  in  the  conditions  of  growth  at  one  time 
D.  9 


130  SUPPOSED  INSTANCES  [OH.  ix 

fostered  the  growth  of  the  first  organism  almost  to  the 
exclusion  of  the  second,  and  at  another  time  fostered  the 
growth  of  the  second  almost  to  the  exclusion  of  the  first. 

If,  after  each  appearance  of  S.  faecalis,  the  strain  had 
been  guaranteed  pure  by  the  method  of  successive  plating 
and  growth  from  a  single  organism,  the  results  obtained 
would  have  had  more  weight. 

It  is  worth  noting  that  the  strains  of  B.  faecalis  al- 
caligenes  used  in  all  these  experiments,  and  supposed  to 
have  been  derived  from  B.  typhosus  in  the  first  place,  showed 
no  tendency  to  revert  to  B.  typhosus.  It  is  also  interesting 
to  compare  these  experiments  with  those  of  Adami,  Abbott  and 
Nicholson  (1899)  -who  obtained  from  B.  coli  grown  in  peri- 
toneal fluid  cocci  which  did  not  ferment  sugars  or  form  indol, 
and  yielded  colonies  which  were  white  and  opaque  and 
resembled  those  of  a  streptococcus.  After  3  passages  through 
the  guineapig  these  cocci  yield  short  bacilli  which  however 
were  still  unable  to  ferment  sugars.  They  state  that  B. 
typhosus  was  modified  in  much  the  same  way  under  similar 
conditions. 

(h)  The  change  from  B.  typhosus  to  B.  faecalis  alcaligenes 
after  growth  in  the  diluted  and  filtered  urine  of  a  typhoid 
carrier. 

(Page  239,  exp.  III.)  This  experiment  was  practically  a 
repetition  of  the  last,  the  same  strain  of  B.  typhosus  being 
used  (laboratory  stock  "R")  but  the  urine  was  that  of  a 
different  typhoid  carrier  "I".  The  result  was  the  same, 
colonies  of  typical  B.  typhosus  being  found  at  first  but  after 
a  month's  interval  colonies  of  a  non-fermenting  and  non- 
agglutinating  coliform  organism  being  alone  found.  To  this 
experiment  the  same  criticism  applies.  19  other  experiments 
were  made  but  with  negative  results. 

Summary.  The  evidence  that  Major  Horrocks  brings 
forward  in  support  of  the  claim  that  in  the  course  of  his 
experiments  transmutation  occurred  is  inconclusive.  He  is 
unable  in  any  case  to  guarantee  as  pure  the  culture  with 
which  he  was  dealing.  He  is  unable  to  exclude  definitely,  in 
some  of  his  experiments,  the  occurrence  of  a  secondary 


CH.  ix]  OF  TRANSMUTATION  131 

invasion  in  the  living  body.  Lastly,  even  if  the  evidence 
established  the  continuity  between  his  original  strains  and 
the  new  ones  he  obtained,  the  changes  may  possibly  have 
been  merely  examples  of  variation  no  greater  in  degree  than 
many  that  have  been  recorded  by  other  observers.  In  other 
words,  the  strains  of  atypical  B.  coli  and  those  closely 
resembling  B.  faecalis  alcaligenes  or  S.  faecalis,  may  con- 
ceivably have  been  variants  of  his  original  strains  of  B. 
typhosus  whose  true  identity  would  have  been  disclosed  by 
more  prolonged  efforts  to  obtain  reversion  or  more  thorough 
investigation  with  regard  to  their  agglutination  reactions. 


^  II.  THE  RELATIONSHIP  BETWEEN  MEMBERS  OF  THE  ENTERITIS 
GROUP  OF  BACILLI — B.  ENTERITIDIS  "  GAERTNER  "  AND  THE 
PARATYPHOID  BACILLUS  OF  THE  "  AERTRYCK  "  OR  "  FLUGGE  " 
TYPE. 

The  question  of  the  specific  character  of  these  organisms 
has  been  much  discussed.  They  are  generally  recognised  as 
distinct  species  but  evidence  has  been  brought  forward  from 
time  to  time  suggesting  that  they  may  be  transmuted  one 
into  the  other. 

A.   Schmitt's  experiments. 

Schmitt  (1911),  for  example,  claims  that  in  certain  experi- 
ments conducted  by  him  strains  of  the  paratyphoid  bacillus 
of  the  Fliigge  type  became  changed  within  the  animal  body 
into  the  Gaertner  type  of  bacillus.  The  details  of  the  experi- 
ments are  briefly  as  follows. 

Experiment  I.  On  July  17th,  21st  and  28th  he  fed  a 
young  calf  on  milk  to  which  had  been  added  (in  amounts 
varying  from  1  to  50  c.c.)  a  broth  culture  of  a  Fliigge  type  of 
organism — without  apparent  effect,  except  that  the  blood 
serum  which  previously  did  not  agglutinate  the  organism  now 
did  so  in  dilutions  of  1  in  35. 

On  August  3rd  the  same  strain  of  organisms  was  injected 
subcutaneously  into  the  calf,  with  the  result  that  the  calf 
became  ill.  An  organism  ("Pgst  I")  was  isolated  from  the 

9—2 


132  SUPPOSED  INSTANCES  [OH.  ix 

calf  s  blood  on  the  same  day.  It  was  found  to  be  agglutinated 
by  the  animal's  blood  serum  in  dilutions  of  1  in  50 — 60 
although,  again,  serum  which  had  been  taken  from  the  calf 
before  the  experiments  began  failed  to  agglutinate  it.  (A 
calf  serum,  immunised  against  a  Gaertner  strain  from  cattle, 
was  on  the  same  day  injected  intravenously  but  only  made 
the  calf  more  ill.)  A  broth  culture  of  this  organism,  Pgst  I, 
was  injected  into  the  same  animal  on  August  7th  and  again 
on  August  10th,  giving  rise  only  to  a  slight  febrile  reaction 
on  each  occasion. 

Experiment  II.  On  August  25th,  the  strain  already 
mentioned  (Pgst  I)  as  having  been  isolated  from  the  first 
calf  s  blood  was  suspended  in  normal  saline  and  sprayed  into 
the  nose  of  another  calf — without  apparent  effect.  On  August 
28th  a  similar  suspension  was  injected  into  the  animal's 
mouth  with  the  result  that  the  calf  became  ill.  An  organism 
(Pgst  II)  was  isolated  from  the  calf  s  blood.  On  September 
4th  a  saline  suspension  of  this  organism  was  injected  intra- 
venously into  the  same  calf,  which  died  a  few  hours  later.  From 
the  blood,  intestines,  muscles  and  bone  marrow  was  obtained 
an  organism  Pgst  III.  The  "  Schluss-serum "  was  found  to 
agglutinate  the  original  Fliigge  type  and  also  the  strains 
Pgst  I,  II,  and  III— in  dilutions  of  1  in  3000—4500. 

The  great  interest  of  the  experiments  lies  in  the  obser- 
vation that  these  later  strains  isolated  from  the  blood  of  the 
calf  were  found  to  correspond  in  their  agglutination  reactions, 
not  with  the  original  Fliigge  type  but  with  the  Gaertner  type 
of  bacillus — a  conclusion  confirmed  by  absorption  tests. 
Schmitt  maintained,  therefore,  that  passage  through  the  calf 
modified  the  agglutination  properties  of  the  human  para- 
typhoid bacillus  ("  Fliigge  ")  so  that  it  came  to  resemble  the 
calf  paratyphoid  bacillus  ("  Gaertner  "). 

Two  possible  fallacies  at  once  suggest  themselves.  The 
type  of  organism  which  made  its  appearance  later  in  the 
experiment  might  have  been  present  as  a  contamination  either 
(a)  in  the  original  strain  or  (b)  in  the  bodies  of  the  animals 
inoculated. 

(a)  As  regards  the  first  alternative,  it  is  conceivable  that 


CH.  ix]  OF  TRANSMUTATION  133 

if  bacilli  of  the  Gaertner  type  were  present  in  the  original  strain, 
but  in  such  scanty  numbers  as  to  escape  detection,  these  few 
bacilli  might  in  the  living  tissues  multiply  with  such  rapidity 
as  to  become  ultimately  the  predominant  organism.  The  pre- 
cautions taken  to  secure  the  purity  of  the  original  culture 
would  presumably  exclude  such  a  source  of  error. 

(6)  In  the  second  place,  bacilli  of  the  later  or  Gaertner 
type  might  conceivably  have  been  present,  before  the  inocu- 
lations were  made,  in  the  bodies  of  the  calves  themselves. 
This  possibility  appears  at  first  sight  to  be  excluded  by  the 
fact  that  the  serum  of  the  calf  before  inoculation  failed  to 
agglutinate  the  organism  recovered  afterwards,  although  the 
serum  after  inoculation  was  able  to  do  so.  This  observation 
is  not,  however,  final. 

Savage  (1907-8)  and  other  observers  have  recorded  the 
presence  of  B.  Gaertner  in  the  intestines  of  healthy  young 
calves.  Its  presence  in  the  intestines  of  the  calves  inoculated 
in  these  experiments  was  not  definitely  excluded. 

The  repeated  administration  in  the  food  of  as  much  as 
50  c.c.  of  a  young  broth  culture  of  a  pathogenic  organism  would 
be  likely  to  cause  an  inflammatory  reaction  in  the  bowel 
and  such  inflammation  might  lead,  not  only  to  an  enormous 
increase  in  numbers  on  the  part  of  any  other  pathogenic 
organism  present,  but  also  to  an  exaltation  in  their  virulence. 
We  have  referred  elsewhere  (vide  p.  80)  to  such  a  sequence  in 
the  case  of  B.  coli  in  the  intestine  during  an  attack  of  typhoid 
fever  and  in  inflammatory  conditions  resulting  from  improper 
food. 

Such  an  increase  both  in  numbers  and  in  virulence  on  the 
part  of  the  organism  we  are  discussing  might  well  pave  the 
way  for  its  invasion  of  the  system  as  a  whole  and  lead  to  its 
appearance  in  the  blood  and  internal  organs,  in  a  manner 
analogous  to  the  invasion  of  the  body  by  saprophytic  organ- 
isms of  heightened  virulence  in  the  cavity  of  an  inflamed 
uterus.  At  the  same  time  the  blood  serum  would  acquire 
agglutination  properties  which  it  did  not  possess  when  the 
bacilli  were  few  in  number,  of  low  virulence  and  restricted  to 
the  lumen  of  the  intestine. 


134  SUPPOSED  INSTANCES  [CH.  ix 

B.  The  experiments  ofMiihlens,  Dahm  and  Filrst. 

These  writers  (1909)  have  recorded  experiments  which 
suggest  a  similar  transmutation.  They  fed  a  large  number  of 
mice  on  meat  which  was  thought  to  have  been  infected 
although  a  preliminary  bacteriological  examination  proved 
negative.  Over  50  per  cent,  of  the  mice  died. 

A  bacteriological  examination  of  the  faeces  of  56  mice  was 
made.  In  20  cases  none  of  the  paratyphoid  group  of  bacilli 
were  detected.  Aertryck's  bacillus  was  found  to  be  present  in 
24  and  Gaertner's  bacillus  in  13  cases. 

These  results  suggest  that  one  type  may  have  arisen  from 
the  other  within  the  animal  body. 

As  in  the  experiments  already  discussed,  two  possibilities 
have  first  to  be  excluded — namely,  the  possibility  of  con- 
tamination (a)  in  the  original  strain  and  (b)  in  the  bodies  of 
the  mice  used  for  the  experiment. 

(a)  All  that  can  be  said  by  way  of  excluding  the  first  of 
these  alternatives  is  that  the  simultaneous  presence  of  both 
the  Aertryck  and  the  Gaertner  type  of  organism  in  infected 
meat  is  contrary  to  experience  and  was  thought  by  other 
writers  (Zwich  and  Weichel,  1910)  to  be  highly  improbable 
in  this  instance. 

(b)  With  regard  to  the  second  alternative,  no  preliminary 
bacteriological  examination  of  the  mice  used  in  the  experi- 
ment was  made.  The  faeces  of  40  control  mice  were  examined 
and  B.  Gaertner  was  discovered  in  one  case  only.  Zwich  and 
Weichel  (1910),  on  the  other  hand,  found  that  out  of  177 
healthy  mice  28  gave  B.  Aertryck  in  the  faeces. 

The  bacteriological  examination  of  the  40  control  mice 
with  a  negative  result  in  39  cases  is,  again,  open  to  the 
criticism  that  paratyphoid  organisms  might  have  been 
actually  present  at  the  time  but  in  such  small  numbers  that 
they  escaped  detection.  Any  such  organisms  present,  in 
equally  small  numbers,  in  the  other  mice  would  find  a  nidus 
for  their  growth  in  the  unhealthy  and  inflamed  condition  of 
the  intestine  which  would  result  from  feeding  the  mice  on 
infected  meat,  while  the  disturbed  functions  of  the  bowel,  by 


CH.  ix]  OF  TRANSMUTATION  135 

hastening  its  evacuation,  would  bring  about  the  subsequent 
appearance  of  these  organisms  in  the  faeces. 

C.   The  author's  experiments. 

The  following  experiments  were  suggested  to  the  writer 
by  Professor  F.  A.  Bainbridge  as  likely  to  throw  some  further 
light  on  this  aspect  of  the  question  and  were  carried  out  at  the 
Lister  Institute  under  his  kind  supervision. 

Experiment  /.  24  healthy  guineapigs  were  chosen  and  12 
of  these  were  confined  in  3  cages.  On  August  13th  one  of  the 
four  guineapigs  in  each  cage  was  removed  and  given  in  its 
food  1  c.c.  of  a  48  hours'  broth  culture  of  B.  enteritidis 
Gaertner.  Every  precaution  was  taken  to  prevent,  as  far  as 
possible,  any  external  contamination  of  the  guineapigs  with 
the  food  and  they  were  then  returned  to  their  respective 
cages.  This  culture  of  B.  Gaertner  was  made  from  a  labo- 
ratory strain  the  agglutination  reactions  of  which  had  been 
repeatedly  tested  and  had  been  found  to  be  constant. 

A  bacteriological  examination  of  the  faeces  of  the  12 
guineapigs  was  made  subsequently  on  three  occasions,  namely 
on  August  23rd,  September  19th  and  October  9th — bacilli  of 
the  paratyphoid  group  being  identified  by  agglutination  tests. 
During  this  period  each  cage  was  kept  separate  from  the 
others  and  none  of  the  guineapigs  were  removed  from  their 
cages  except  for  the  necessary  examination  on  the  three 
dates  mentioned.  Up  to  the  time  of  the  first  examination  on 
August  23rd  all  the  guineapigs  remained  well,  but  subse- 
quently three  of  them  died  and  the  remainder  exhibited 
varying  degrees  of  malaise  and  intestinal  disturbance.  The 
following  scheme  shows  the  type  of  organism  found  in  the 
faeces  at  each  examination. 

N.B.  Guineapig  No.  1  in  each  cage  was  given  1  c.c.  of  a 
broth  culture  of  B.  enteritidis  Gaertner  on  August  13th.  "  0  " 
indicates  that  neither  the  Gaertner  nor  the  Aertryck  type  of 
organism  was  isolated. 

The  faeces  of  the  remaining  12  guineapigs,  which  had 
been  kept  quite  apart  from  the  others,  were  carefully  investi- 


136 


SUPPOSED  INSTANCES 


[CH.  IX 


gated  at  the  date  of  the  first  examination  (August  23rd). 
Gaertner's  bacillus  was  not  found  in  any  case  and  Aertryck's 
bacillus  in  one  case  only. 


Cage 

Guineapig 

1st  examination 
August  23rd 

2nd  examination 
Sept.  19th 

3rd  examination 
Oct.  9th 

A 

No.  1 
No.  2 
No.  3 

No.  4 

Aertryck 
0 
Gaertner 

Gaertner 

Dead 
Aertryck 
Dead 
P.  M.  Gaetner 
Aertryck 

Gaertner 
Gaertner 

B 

No.  1 
No.  2 
No.  3 
No.  4 

Aertryck 
Aertryck 
Gaertner 
0 

0 
0 
Dead 
0 

0 
0 

(no  faeces) 

C 

No.  1 
No.  2 
No.  3 

No.  4 

Gaertner 
Aertryck 
Aertryck  and 
Gaertner 
0 

Aertryck 
Gaertner 
(no  faeces) 

(no  faeces) 

0 
0 

These  experiments  appeared  to  lend  further  support  to 
the  theory  that  the  two  types  of  organisms  were  capable  of 
transmutation.  It  is  necessary,  however,  again  to  emphasise 
the  fact  that  a  negative  result  in  the  case  of  all  but  one  of  the 
animals  examined  as  a  control,  does  not  prove  the  absence  of 
organisms — it  only  proves  their  scarcity.  A  subsequent  in- 
crease in  their  numbers  might  at  once  have  revealed  their 
presence.  Such  an  increase  might  be  apparent  only,  due  to 
a  simple  disturbance  of  the  functions  of  the  bowel,  such  as 
diarrhoea,  which  would  dislodge  the  organism  from  its  usual 
habitat,  carry  it  to  a  lower  part  of  the  bowel  and  hasten  its 
evacuation.  The  increase  in  numbers  might,  on  the  other  hand, 
be  a  real  one,  brought  about  by  a  lowered  vitality  of  the 
body  as  a  whole  and  local  inflammatory  changes.  It  is  to  such 
factors  that  we  attribute  the  enormous  number  of  B.  coli 
found  in  the  stools  of  patients  suffering  from  cholera. 

All  these  factors  were,  no  doubt,  operative  in  the  case  of 
the  three  guineapigs  which  were  actually  fed  with  the  culture 
of  B.  Gaertner  and  may  explain  the  subsequent  discovery  of 


CH.  ix]  OF  TRANSMUTATION  137 

B.  Aertryck  in  the  faeces  of  all  of  them.  The  remaining 
guineapigs  may  have  been  infected  by  B.  Aertryck  from  the 
faeces  of  these,  at  one  stage  or  another,  owing  to  their  food 
becoming  contaminated.  The  same  factors  would  explain,  in 
their  case,  the  later  appearance  of  B.  Gaertner. 

It  is,  however,  to  be  noted  that  at  the  1st  examination 
(August  23rd)  of  the  guineapigs  in  cages  A  and  B,  while  both 
those  which  had  been  fed  with  the  Gaertner  culture  were 
passing  B.  Aertryck  in  their  faeces,  three  of  the  six  animals 
which  had  not  been  fed  with  the  Gaertner  culture  were 
passing  B.  Gaertner.  To  explain  this  on  the  grounds  that  the 
food  of  these  three  had  been  contaminated  by  the  faeces  of 
the  Gaertner  fed  guineapigs,  we  must  assume  that  the  latter 
had,  at  some  time  previous  to  the  examination,  been  also 
passing  B.  Gaertner  in  their  faeces  and  that  this  organism 
had  only  later  given  place  to  B.  Aertryck. 

That  the  factors  we  have  been  discussing  do  actually  lead 
to  the  detection  in  the  faeces  of  organisms  which  previously 
did  not  appear  to  be  present,  the  writer  endeavoured  to  prove 
by  further  experiment. 

Experiment  II.  On  August  25th  six  apparently  healthy 
guineapigs  were  chosen  and  labelled  Nos.  1  to  6.  A  frag- 
ment of  the  faeces  from  each  guineapig  was  shaken  up  in 
malachite  green  broth  and  after  incubation  the  latter  was 
plated  out,  two  sterile  plates  being  used  for  each  guineapig. 

Both  plates  from  No.  3  showed  white  colonies.  A  culture 
from  these  colonies  was  grown  in  broth  for  48  hours  and  then 
passed  through  the  sugars  by  which  means  it  was  identified 
as  B.  proteus.  In  the  case  of  the  remainder  the  five  pairs  of 
plates  all  proved  to  be  sterile. 

The  guineapigs  were  then  for  several  days  fed  on  green 
vegetables  and  bran  soaked  in  castor  oil — a  diet  designedly 
unwholesome  and  calculated  to  make  the  animals  ill  and  also 
to  set  up  some  intestinal  catarrh.  On  August  30th  a  fragment 
of  faeces  from  each  guineapig  was  again  shaken  up  in 
malachite  green  broth  and  this,  after  incubation,  plated  out 
as  before  on  two  sterile  plates. 

In  the  case  of  No.  1  and  No.  5  no  growth  was  apparent  in 


138  SUPPOSED  INSTANCES  [CH.  ix 

the  tubes  and  both  pairs  of  plates  remained  sterile.  In  the 
other  four  tubes  growth  took  place  with  gas  formation  and 
the  four  pairs  of  plates  all  showed  colonies.  Broth  cultures 
were  made  from  these  colonies  and  yielded  strains  which  gave 
the  sugar  reactions  of  B.  proteus. 

The  writer  failed  to  demonstrate  the  presence  in  any  case  of 
organisms  of  the  paratyphoid  group.  The  experiment  however 
was  successful  in  demonstrating  that  bacteria  which  failed  to 
give  evidence  of  their  presence  in  the  faeces  of  a  healthy 
guineapig  might  make  their  appearance  in  the  faeces  of  the 
same  animal  after  it  had  been  given  for  a  few  days  unwhole- 
some and  irritating  food. 

This  conclusion  lends  weight  to  the  suggestion  already 
made  that  the  results  obtained  by  Schmitt,  and  also  by 
Miihlens,  Dahm  and  Fiirst,  might  possibly  be  explained  by  the 
presence  of  a  secondary  invader. 

Their  experiments  are,  in  both  instances,  open  to  one 
further  criticism.  The  identification  of  the  paratyphoid  or- 
ganisms was  made  to  depend  solely  upon  their  agglutination 
reactions.  If  it  is  admitted  that  the  power  to  form  and  to 
absorb  specific  agglutinins  on  the  part  of  an  organism  is 
subject  to  variation  it  must  be  recognised  that  such  tests 
alone  are  insufficient  to  establish  the  identity  of  the  organism. 
In  other  words,  it  is  within  the  bounds  of  possibility  that  only 
one  type  of  organism  was  actually  present,  but  that  its  agglu- 
tination properties  varied. 

Such  a  contingency  would  be  likely  to  arise  in  the  case  of 
two  organisms  so  closely  allied  as  B.  Aertryck  and  B.  Gaertner. 
An  elaborate  investigation  into  the  agglutination  properties 
of  these  two  organisms  was  conducted  by  Sobernheim  and 
Seligmann  (1910).  Pure  colonies  of  numerous  strains  were 
secured  by  the  Indian  ink  method.  They  found  that  colonies 
derived  from  the  same  strain  and  growing  side  by  side  differed 
in  their  agglutination  reactions.  The  same  strain  differed  at 
different  times.  The  agglutination  reactions,  in  some  instances, 
became  altered  after  passage  through  the  mouse  and  after  a 
culture  had  been  heated.  In  some  instances  the  injection  of 
living  bacilli  yielded  a  serum  which  was  much  more  variable 


CH.  ix]  OF  TRANSMUTATION  139 

in  its  agglutinative  powers  than  a  serum  obtained  by  means 
of  a  dead  culture.  Some  strains  gave  doubtful  reactions.  The 
power  of  the  same  strain  to  form  agglutinins  and  to  bind 
agglutinins  appeared  in  some  cases  to  differ.  They  therefore 
concluded  that  the  agglutination  reactions  did  not  constitute 
a  specific  test. 

We  may  interpret  these  results  in  one  of  two  ways.  We 
may  decline  to  recognise  the  two  types  as  representing  dis- 
tinct species  ;  or  we  may  continue  to  regard  them  as  distinct 
species  and  acknowledge  that  their  agglutination  properties 
are  liable  to  variation.  In  either  case  the  experiments 
quoted  are  deprived  of  all  significance  as  examples  of 
transmutation. 


CHAPTER  X 

SUMMARY 

IT  will  be  evident  from  the  foregoing  pages  that  practically 
every  character  of  bacteria  is  liable  to  vary  at  different  times 
and  under  different  conditions.  These  variations  are  of  two 
kinds,  spontaneous  or  "intrinsic" — that  is  to  say  due  to 
tendencies  inherent  in  the  organism  itself — and  impressed 
as  a  result  of  external  influences.  These  modifying  influences 
have  been  enumerated  (Chapter  II)  and  examples  given  of 
the  variations  they  produce. 

In  many  cases  an  organism  may  appear  to  vary  although 
no  variation  actually  takes  place,  and  in  other  cases  what 
appears  to  be  a  "spontaneous"  variation  is  actually  an  "im- 
pressed" variation  due  to  external  influences  which  have  not 
been  recognised  by  the  observer.  These  various  sources  of 
error  have  been  enumerated  and  discussed  in  Chapter  III. 

A  tendency  to  vary  in  a  particular  way — either  spontane- 
ously or  in  response  to  external  stimuli — may  be  so  charac- 
teristic of  a  certain  organism  as  to  be  in  itself  almost  specific 
in  character,  and  so  far  from  confusing  its  identity  may 
actually  make  this  more  apparent.  The  pleomorphism  of 
B.  diphtheriae,  the  tendency  of  S.  scarlatinae  to  assume 
a  bacillary  shape,  the  tendency  of  B.  paratyphoid  B  to  form 
papillae  on  raffinose  agar,  will  serve  as  examples. 

No  single  property  of  bacteria  can  be  regarded  as  specific 
nor  does  the  occurrence  of  variation  in  respect  to  any  one 
quality  or  function,  or  to  several  of  them  simultaneously, 
necessarily  imply  a  loss  of  specific  character  on  the  part  of  the 
organism  concerned.  This  is  well  illustrated  by  the  mor- 
phology of  bacteria.  A  certain  appearance  may  be  spoken 
of  as  "characteristic."  This  does  not  mean  that  it  is  in- 
variable but  merely  that  the  organism  shows  a  tendency  to 


CH.  x]  SUMMARY  141 

present  such  an  appearance  rather  than  another.  B.  coli  in 
the  peritoneal  cavity  in  the  case  of  ascites  may  take  the  form  of 
a  diplococcus;  in  milk  or  in  urine  it  may  develop  into  a 
dense  network  of  branching  filaments  resembling  B.anihracis, 
but  these  changes  in  form  do  not  imply  any  obliteration  of  the 
specific  character  of  the  organism  itself. 

We  have  already  referred  in  this  connection  (vide  p.  38) 
to  the  analogy  of  a  regiment  of  soldiers  at  manoeuvres  and 
a  mass  meeting  of  miners  at  the  pithead.  The  various  military 
formations  assumed  by  the  first  are  as  characteristic  as  the 
concentrically  arranged  crowd  formed  by  the  second — so  much 
so  that  an  observer  at  a  distance  might  from  the  appearance 
of  these  "zoogleic  forms"  state  with  confidence  the  character 
of  the  units  composing  them  although  too  far  away  to  identify 
the  latter.  A  crowd  of  pitmen  on  strike  might,  however,  march 
in  military  formation  and  a  regiment  of  soldiers  at  a  boxing 
match  take  the  form  of  a  crowd  concentrically  arranged — each 
reproducing,  that  is  to  say,  the  appearance  regarded  as  typical 
of  the  other.  This  would  not  indicate  that  the  pitmen  were 
changing  into  soldiers  or  the  soldiers  into  pitmen.  It  is  true, 
nevertheless,  that  the  arrangement  most  frequently  observed 
in  one  or  the  other  case  does  indicate  a  tendency  on  the  part 
of  the  individual  unit  and  may,  therefore,  afford  a  clue  to 
its  identification.  The  behaviour  of  a  civilian  under  certain 
circumstances  may  furnish  evidence  of  a  military  training  and 
deserters  from  the  colours  are  not  infrequently  recognised  by 
such  means.  In  a  similar  way,  the  occasional  assumption  by 
the  bacillus  of  diphtheria  of  clubbed  and  branched  forms, 
while  helping  us  to  identify  it,  also  provides  us  with  a  clue  to 
its  mycelial  ancestry  (Kanthack  and  Andrewes,  1905). 

Many  of  the  variations  exhibited  by  bacteria  do  in  fact, 
represent  steps  in  the  evolutionary  process  by  which,  in 
the  past,  they  have  become  differentiated — the  individual 
organisms  living  over  again,  as  it  were,  the  life  history  of  the 
race.  This  would  appear  to  be  the  explanation  of  many 
variations  in  morphology  (Chapter  IV).  Others  again  repre- 
sent the  advance  along  new  lines  of  this  same  evolutionary 
process,  leading  to  further  specialisation  and  differentiation. 


142  SUMMARY  [OH.  x 

This  aspect  of  the  subject  has  been  considered  at  length  in 
the  sections  dealing  with  Fermenting  Power  and  Virulence 
(Chapters  Y  and  VI). 

Since  we  have  no  absolute  criterion  as  to  what  constitutes 
a  "species"  amongst  bacteria,  dissimilarity  in  the  several 
characters  they  present  is  our  sole  guide  to  classification. 
In  other  words  the  distinction  between  a  "variety"  and  a 
"species"  depends  simply  on  less  or  greater  divergence  in 
character.  The  difference,  therefore,  between  variation  and 
transmutation  is  one  of  degree  only ;  or,  looking  at  the  matter 
from  another  standpoint,  we  may  say  that  the  same  degree 
of  deviation  from  type  may  be  interpreted  in  one  case  as 
variation  and  in  another  as  transmutation.  This  will  be  readily 
understood  if  it  be  borne  in  mind  that  the  various  types  or 
"species"  of  bacteria  which  we  are  able  to  distinguish  have 
developed  from  a  common  stock.  In  the  case  of  some  of  them 
the  differentiation  dates  from  a  remote  past  and  the  specific 
characters  are  comparatively  fixed.  In  the  case  of  others 
differentiation  is  of  more  recent  date  and  the  newly  acquired 
characters  are  less  permanent  and  "reversion"  in  one  or  other 
character  is  more  frequent.  In  yet  a  third  class — the  groups 
of  closely  allied  organisms — the  gradual  process  of  differentia- 
tion is  only  now  taking  place  and  it  is  not  yet  clear  which 
characters  are  of  specific  value.  During  the  process  of  evolu- 
tion, in  all  its  stages,  there  is  a  tendency  shown  on  the  part 
of  the  organism  to  revert  towards  the  original  type.  Such 
reversion  in  one  or  more  characters,  although  of  no  greater 
significance  in  the  case  of  one  class  or  another  of  the  three 
we  have  described,  is  likely  to  be  differently  interpreted.  If 
differentiation  is  well  advanced,  a  partial  reversion  in  character 
will  merely  present  itself  as  an  unimportant  variation.  If 
differentiation  has  not  progressed  very  far,  a  reversion  no 
greater  in  degree  may  confuse  the  identity  of  the  organism 
concerned  sufficiently  to  suggest  the  possibility  that  trans- 
mutation has  occurred.  If  the  process  of  differentiation  is 
still  incomplete,  a  reversion  in  character  even  smaller  in  degree 
may  entirely  obliterate  the  faint  lines  of  division  that  we  have 
been  able  to  trace  out.  The  error  of  assuming  too  hastily  that 


CH.  x]  SUMMARY  143 

transmutation  has  occurred  will  be  prevented  by  a  proper 
consideration  of,  firstly,  the  biological  characters  of  the 
organism  in  question  as  a  whole  and,  secondly,  the  question 
of  the  stability  of  the  characters  which  distinguish  it. 

With  regard  to  the  first  of  these  questions,  we  have  shown 
in  the  sections  dealing  with  Morphology,  Fermenting  power, 
Virulence  and  Pathogenesis  (Chapters  IV- VII)  the  danger  of 
relying  upon  any  one  of  these  characters  alone  for  the  purpose 
of  identification  or  of  classification. 

In  the  case  of  widely  divergent  types  a  single  character 
may  sometimes  suffice  to  distinguish  one  organism  from 
another  but  even  in  such  a  case,  if  that  character  is  liable 
under  any  circumstances  to  variation,  it  obviously  cannot  be 
trusted  as  an  infallible  guide. 

The  pathologist  is  in  the  same  boat,  in  this  respect,  with 
the  ethnologist.  Certain  "race  groups,"  e.g.  the  Teutonic,  the 
Mongolian,  and  the  Negroid,  though  conceivably  derived  from 
a  common  anthropoid  stock,  are  sufficiently  differentiated  to 
be  readily  distinguished  by  a  single  character.  For  example, 
the  flaxen  hair  of  the  German,  the  matted  black  hair  of  the 
Negro  and  the  straight  black  hair  of  the  Jap  are  sufficiently 
characteristic  of  their  respective  race-groups.  Such  a  dis- 
tinction, however,  breaks  down  between  the  races  within 
the  groups  themselves  and  other  characters  must  then  be 
considered  in  addition.  In  some  cases,  again,  the  process  of 
differentiation  is  still  incomplete,  individuals  approximating 
now  to  one  and  now  to  another  recognised  type,  and  a  con- 
sideration of  all  the  characters  may  still  leave  the  observer 
in  doubt  as  to  the  correct  classification. 

The  ethnologist  has  learnt,  moreover,  that  certain  char- 
acteristics are  not  to  be  regarded  as  racial  in  character.  For 
example — to  return  to  our  previous  illustration — the  pigtail 
of  the  Chinaman  and  the  shaven  poll  of  the  Thibetan  priest, 
the  flowing  locks  of  an  Italian  impressario  and  the  tonsured 
crown  of  a  Romish  monk,  are  not  racial  characters  at  all  but 
artificial  modifications.  They  do,  however,  signify  a  certain 
environment  and  training  and  this  is  precisely  the  case  with 
many  of  the  variations  which  the  pathologist  meets  with 


144  SUMMARY  [CH.  x 

amongst  bacteria.  In  other  words,  a  study  of  such  variations 
in  a  given  case  may  afford  valuable  and  trustworthy  informa- 
tion as  to  the  source  from  which  the  particular  strain  of 
organisms  has  been  derived. 

This  subject  would  repay  further  investigation.  One 
or  two  instances  may  be  given  here  to  demonstrate  its  im- 
portance. 

Rosenow  (1912-13)  found  that  the  ordinary  streptococcus 
pyogenes,  if  grown  in  unheated  milk,  became  modified  in  its 
morphology,  its  cultural  properties  and  its  virulence.  He  had 
previously  isolated  from  several  cases  of  epidemic  sore  throat 
a  streptococcus  which  possessed  precisely  similar  modifications 
in  character.  The  epidemic  had  been  recognised  as  "milk- 
borne"  but,  had  its  origin  been  in  doubt,  the  unusual  char- 
acters of  the  organism  concerned  would  obviously  have  pro- 
vided a  clue. 

Ohlmacher  (1902)  isolated  branching  filamentous  forms  of 
B.  coli  from  the  heart's  blood  in  a  case  of  septicaemia.  He 
quotes  various  observations  to  the  effect  that  residence  in  the 
biliary  passages  develops  this  unusual  morphology  in  B.  coli, 
and  he  therefore  considers  that  the  original  source  of  the 
systemic  infection  in  this  case  was  in  the  region  of  the  gall 
bladder  or  bile  ducts. 

Moreover  the  degree  to  which  the  modifications  persist  on 
subculture  is  a  measure  of  the  time  during  which  the  organism 
was  subjected  to  the  modifying  influence.  This  brings  one  to 
the  second  question,  the  stability  of  the  variations  produced. 

Remarkable  differences  are  to  be  observed  in  the  degree 
of  permanence  exhibited  by  a  variation  in  different  cases  and 
it  is  difficult  to  decide  upon  what  factors  these  differences 
depend. 

We  have  already  referred  to  the  fact  that  variations  may 
be  either  "spontaneous"  in  character  or  "impressed"  upon 
the  organism  by  external  agencies.  Spontaneous  variations 
may  be  of  several  kinds  and  the  nature  of  the  variation  may 
itself  decide  its  degree  of  permanence. 

1.  Some  variations  represent  an  early  stage  in  the  life 
history  or  are  due  to  imperfect  development,  and  are  seen 


CH.  x]  SUMMARY  145 

in  young  or  backward  cultures.  We  have  spoken  of  the  atypical 
morphology  of  a  young  culture  of  the  Klebs-Loeffler  bacillus, 
which  renders  it  difficult  to  distinguish  it  from  Hofmann's 
bacillus  (vide  p.  42),  and  of  its  inability  to  ferment  glycerin  and 
lactose  (vide  p.  55).  Such  differences  are  comparable  to  the 
juvenile  features  and  unskilled  hands  of  a  class  of  schoolboys 
and  tend  to  disappear  of  their  own  accord  as  the  strain  grows 
and  develops. 

2.  Other  variations  represent  senile  changes  or  are  due  to 
lowered  vitality,  and  are  seen  in  old  or  worn  out  strains.  The 
loss  of  motility,  or  of  pigment  production,  in  an  old  culture 
will  serve  as  an  example.    Such  variations  are  comparable  to 
the  slow  steps  and  grey  hairs  that  characterise  a  party  of  old 
men  and  will  tend  to  become  more  and  more  developed  unless 
some  external  influence  intervenes  and,  by  effecting  a  radical 
change  in  the  conditions  of  growth,  contrives  to  rejuvenate 
the  strain. 

3.  Others  again  are  degenerative  in  character  or  are  due 
to  atavistic  tendencies — such  as,  for  example,  the  appearance 
of  branched  and  clubbed  forms  of  the  tubercle  bacillus.   These 
variations  are  comparable  to  some  forms  of  mental  impairment 
in  a  family,  or  to  defects  such  as  harelip.  They  may  be  passed 
on  from  father  to  son  and  so  persist,  or  they  may  disappear, 
but  in  the  latter  case  they  tend  to  recur  in  a  later  generation. 

4.  Others,  finally,  are  evolutionary  in  character  and  repre- 
sent a  higher  specialisation  on  the  part  of  the  organism — such 
as,  for  instance,  the  development  by  B.  typhosm,  after  a  long 
training,  of  power  to  ferment  lactose,  or  the  acquisition  on 
the  part  of  a  feebly  pathogenic  organism  of  the  quality  of 
extreme  virulence.    Such  changes  are  analogous  to  the  de- 
velopment of  a  national  genius  for  literature  or  conquest. 
The  more  highly  specialised  a  function  is  the  more  easily  does 
it  become  deranged  and  a  character,  therefore,  of  this  kind,  is 
readily  lost.    For  example,  however  permanent  other  newly 
acquired  characters  in  bacteria  may  appear  to  be,  variation 
in  the  direction  of  increased  virulence  seldom  is  so  and  almost 
invariably  proves  unstable. 

It  is  easy  to  see  how,  in  every  one  of  the  four  classes  we 

D.  10 


146  SUMMARY  [OH.  x 

have  mentioned,  two  or  more  variations  may  be  constantly 
associated.  In  some  cases  the  association  is  explained  by  the 
fact  that  both  variations  are  due  to  lowered  vitality.  For 
example,  the  loss  of  power  to  produce  pigment  may  be 
associated  with  the  loss  of  power  to  liquefy  gelatin  or  to  grow 
on  certain  not  very  favourable  media — all  these  functions 
being  dependent  upon  the  vitality  of  the  organism. 

Again,  the  evolution  or  higher  specialisation  of  an  organism 
may  involve  simultaneous  modification  in  two  or  more  direc- 
tions. These  modifications  may  all  represent  a  casting  off  of 
saprophytic  characters  by  the  organism  in  question  on  its 
entry  upon  a  parasitic  career.  For  example,  a  saprophyte 
may  derive  its  vital  energy  from  the  sunlight  by  means  of  a 
pigment,  comparable  to  the  chlorophyll  of  a  vegetable  cell,  or 
from  carbohydrate  food  through  its  ability  to  ferment  it.  When 
it  becomes  parasitic,  and  in  many  cases  pathogenic,  it  is  cut 
off  from  sunlight  and  must  subsist  on  the  body  fluids.  We  may 
find  therefore  that  the  acquirement  of  virulence  is  associated 
with  the  loss  of  power  to  form  pigment  and  to  ferment  sugars. 

In  the  same  way,  the  constant  association  between  two 
different  variations  may  be  due  to  the  fact  that  the  young 
strains  which  show  them  have  not  developed  their  adult  powers, 
or  to  the  fact  that  the  variations  are  both  signs  of  degeneracy 
or  atavism. 

"Impressed"  variations  show  even  greater  differences  in 
their  degree  of  permanence.  In  some  cases  a  variation  is  only 
maintained  while  the  influence  which  caused  it  continues  to 
act.  In  others  the  variation  persists  for  a  shorter  or  longer 
period  after  that  influence  is  withdrawn.  In  others  again  the 
variation  is  apparently  permanent  and  persists  under  normal 
conditions  of  growth  indefinitely.  We  use  the  expression 
"apparently  permanent"  for  it  is  impossible  in  any  case  to 
guarantee  the  permanence  of  the  characters  exhibited  by  a 
strain  of  bacteria.  This  has  been  shown  both  by  observation 
and  by  experiment.  Mention  has  been  made  elsewhere  (vide 
p.  14)  of  a  strain  of  bacteria  which  after  nine  years'  cultivation 
lost  its  power  to  ferment  maltose,  and  of  another  strain  which 
after  five  years  cultivation  lost  its  power  to  produce  pigment. 


CH.  x]  SUMMARY  147 

Twort  found  that  B.  typhosus  grown  in  a  lactose  medium 
retained  its  character  as  a  non-fermenter  of  lactose  for  two 
years  before  variation  occurred.  Eyre  and  Washbourn  found 
that  to  raise  a  particular  strain  of  an  avirulent  saprophytic 
pneumococcus  to  full  virulence  by  animal  passage,  no  less  than 
fifty-three  successive  inoculations  were  required.  Characters 
which  persisted  for  periods  of  two,  five  and  nine  years,  and 
withstood  a  series  of  over  fifty  passages  through  an  animal 
body,  might  well  have  been  regarded  as  "permanent."  They 
were,  however,  only  "apparently"  so. 

Certain  principles  which  govern  the  stability  of  impressed 
variations  can,  however,  be  discerned. 

1.  The  variation  may  affect  all  the  members  of  a  strain 
or  only  certain  of  them.  In  the  latter  case  an  apparent 
reversion  is  obviously  more  likely  to  occur.  The  rapidity  with 
which  this  apparent  reversion  takes  place  will  depend  upon 
the  comparative  rate  of  growth  of  the  unaltered  organisms 
and  the  variants.  If  the  new  character  is  of  advantage  to  the 
organism  it  will  enable  the  variants  to  multiply  more  quickly 
and  they  will  gradually  get  the  upper  hand.  Apparent  re- 
version will  not  take  place  as  long  as  the  new  character 
continues  to  confer  an  advantage  upon  its  possessors  but  when 
this  ceases  to  be  the  case  the  organisms  possessing  the  new 
character  may  disappear  and  reversion  to  the  original  type 
appear  to  take  place.  For  example,  the  acquirement  of  viru- 
lence by  some  members  of  a  non-virulent  or  feebly-virulent 
strain,  when  this  is  injected  into  the  living  body,  gives  these 
variants  an  advantage  as  long  as  they  are  in  the  body.  If  the 
mixed  strain  is  grown  on  artificial  media  the  advantage  is 
done  away  with  and  the  unaltered  bacteria,  other  things  being 
equal,  have  now  as  good  a  chance  as  the  variants  of  increasing 
their  numbers  and  the  variants  may  disappear. 

We  have  used  the  expression  "  apparent  reversion  "  for  it 
is  evident  that,  unless  every  member  of  a  strain  acquires  the 
new  character,  the  loss  of  that  new  character  by  the  strain  may 
be  brought  about  by  the  dying  out  of  the  variants  without  a 
single  organism  having  actually  "  reverted."  This  fallacy  can 
be  readily  excluded  if  care  be  taken  at  each  step  to  ensure  that 

10—2 


148  SUMMARY  [CH.  x 

the  strain  of  bacteria  under  observation  is  a  pure  one — that 
is  to  say,  derived  from  a  single  organism  by  the  methods  sug- 
gested by  Barber  and  others. 

2.  The  more  readily  a  new  character  is  "impressed  "  on  an 
organism  the  longer  it  is  retained — conversely,  the  more  slowly 
and  reluctantly  an  organism  takes  on  a  new  character  the  more 
easily  is  that  character  lost.  The  behaviour  of  B.  typhosus  is 
a  good  illustration.   This  organism  can  be  trained  to  ferment 
dulcite  in  a  few  days  and  will  then  retain  the  power  for  many 
weeks  in  the  absence  of  that  sugar.   It  cannot  be  trained  to 
ferment  lactose  in  less  than  two  years  and  then  loses  the  power 
in  a  few  days  if  the  lactose  is  withdrawn. 

It  must  be  understood  that  we  are  here  speaking  of  "  im- 
pressed "  variations.  In  the  case  of  "  spontaneous  "  variations 
the  reverse  holds  true.  Bacteria  which  vary  spontaneously 
with  great  readiness  often  revert  with  equal  facility,  while 
those  that  are  tenacious  of  their  normal  characters  often 
prove  tenacious  of  any  new  character  they  may  spontaneously 
develop. 

3.  The  longer  an  organism  which  has  undergone  a  varia- 
tion continues  to  be  exposed  to  the  influence  which  caused  it, 
the  longer  will  the  variation  persist  after  that  influence  has 
been  withdrawn. 

For  example,  Rosenow  (1912-13)  isolated  a  streptococcus, 
from  a  number  of  cases  of  general  infection,  possessing  unusual 
morphological  and  cultural  characters  which,  however,  showed 
reversion  on  cultivation  outside  the  body.  He  found  that 
strains  isolated  from  the  peritoneal  exudate  and  blood  at  a 
later  stage  in  the  disease  showed  these  modifications  in  char- 
acter to  a  greater  degree  than  those  isolated  earlier  in  the 
attack. 

The  behaviour  of  B.  typhosus  again  illustrates  this  point. 
If  a  strain  is  grown  on  dulcite  medium  it  acquires,  in  a  few 
days,  the  power  of  fermenting  that  sugar  and  this  power  is 
retained  for  some  weeks  after  the  strain  has  been  removed 
from  the  dulcite  medium.  Reversion  then  occurs  and  the 
power  is  lost.  If  however  the  strain  is  grown  on  a  dulcite 
medium  continuously  for  three  months  the  power  to  ferment 


CH.  x]  SUMMARY  149 

dulcite  is  found  to  persist  afterwards,  on  ordinary  media, 
"  permanently." 

This  observation,  based  on  the  results  of  laboratory  experi- 
ments, provides  a  clue,  as  Adami  observes,  to  the  nature  of  the 
process  by  which  new  races  of  bacteria  are  developed.  In  the 
laboratory  organisms  can  be  exposed  to  certain  modifying 
influences  for  many  months  or  even  years  and  the  new  char- 
acters developed  by  such  means  are  found  to  persist  for  long 
periods  before  reversion  takes  place.  In  nature  agencies  which 
possess  the  power  of  modifying  the  characters  of  bacteria  may 
exert  their  influence  for  an  indefinite  period  and  the  process 
of  reversion  in  this  case  may  be  indefinitely  postponed.  In 
other  words  the  new  characters  developed  may  appear  to  be 
permanent.  A  variant,  however,  may  retain  its  new  characters 
indefinitely  and  show  no  tendency  whatever  to  revert  under 
ordinary  conditions  of  growth  and  yet  it  may  still  be  capable 
of  reverting  immediately  under  suitable  conditions.  Examples 
of  this  are  common  in  the  laboratory  and  may  be  found  in 
nature.  Laurent  describes  a  decolourised  strain  of  B.  ruber 
which  was  grown  for  12  months  at  a  temperature  of  25-35°  C., 
being  subcultured  32  times  in  this  period,  without  once  show- 
ing any  trace  of  pigment.  On  lowering  the  temperature  to  18°C. 
pigmentation  at  once  reappeared.  Again,  the  diphtheria  ba- 
cillus is  far  removed  from  its  mycelial  ancestry  but  under 
suitable  conditions  will  still  display  a  partial  reversion  to  a 
mycelial  structure.  We  do  not  on  this  account  deny  the  title 
of  "  species  "  to  the  diphtheria  bacillus,  for  we  recognise  that 
the  idea  of  absolute  permanence  in  character  is  not  essential 
to  our  conception  of  a  species  in  the  case  of  bacteria.  It  is  not 
permanence  in  character  but  the  degree  of  resistance  to  al- 
teration in  character  displayed  by  an  organism  that  determines 
our  opinion  of  its  specific  nature.  In  spite  of  the  many  minor 
variations  they  display  there  is  exhibited  by  most  species  of 
bacteria  a  resistance  to  modification — a  "  vis  inertia  " — which 
constitutes  true  racial  stability. 

We  have  seen,  then,  that  the  difference  between  variation 
and  transmutation  is  one  of  degree  alone.  It  is  a  question  of 
the  extent  of  the  modification  and  the  degree  of  permanence 


150  SUMMARY  [CH.  x 

it  exhibits.  It  is  no  less  true  that  the  process  of  transmutation 
only  differs  in  degree  from  the  process  of  evolution.  Here  it 
is  a  question  of  the  rapidity  of  the  change. 

Let  us  take  by  way  of  illustration  the  case  of  a  family  of 
ancient  lineage,  the  members  of  which  hold  high  office  in  the 
State  and  are  remarkable  for  their  wealth  and  erudition. 
Such  a  family  may  have  sprung  500  years  ago  from  humble 
origin  and,  while  the  fortunes  of  one  branch  have  steadily 
prospered  and  successive  generations  have  gradually  acquired 
fame  and  amassed  wealth,  the  original  yeoman  stock  from 
which  it  sprang  has  continued  to  be  represented  throughout 
the  centuries,  in  some  corner  of  the  kingdom,  by  men  chiefly 
remarkable  for  their  deficiency  in  the  riches  and  learning  and 
reputation  for  which  the  others  are  distinguished.  It  is  con- 
ceivable that  a  son  of  the  older  and  less  distinguished  branch 
of  the  family,  seizing  a  favourable  opportunity,  might,  by  the 
exercise  of  the  same  faculties  of  industry  and  thrift  displayed 
by  the  others,  raise  himself  in  the  space  of  a  single  life-time  to 
a  position  of  wealth  and  power  equal  to  theirs.  We  can  trace 
the  steps  by  which,  in  the  course  of  time,  a  virulent  and  highly 
specialised  race  of  bacteria  has  been  evolved  from  a  less 
virulent  and  less  highly  organised  race.  We  find  the  two  races 
living  still  side  by  side.  The  question  arises  whether  it  is  pos- 
sible under  unusually  favourable  conditions  for  the  process  of 
adaptation  and  specialisation  to  take  place  with  such  rapidity 
as  to  suggest  a  sudden  transmutation. 

The  conversion  of  the  saprophytic  pneumococcus  into  the 
parasitic  pneumococcus  by  Eyre,  Leatham  and  Washburn 
(vide  p.  115)  appears  to  offer  an  example.  These  observers 
describe  the  virulent  parasitic  pneumococcus  as  requiring  for 
its  growth  a  certain  reaction  and  temperature  and  particular 
media  (blood  agar);  it  would  not  grow  if  the  reaction  were 
even  faintly  acid  or  at  a  temperature  much  below  37°  C.  and 
rapidly  died  out  on  agar  or  in  broth.  It  would  not  liquefy 
gelatin  and  in  broth  formed  a  dust-like  deposit.  The  avirulent 
saprophytic  variety,  on  the  other  hand,  grew  luxuriantly  at 
temperatures  ranging  from  37°  to  20°  C.,  on  agar,  gelatin, 
potato  or  in  broth,  whether  acid  or  alkaline,  slowly  liquefying 


CH.  x]  SUMMARY  151 

gelatin,  producing  a  uniform  turbidity  in  broth,  and  it  retained 
its  vitality  for  many  months ;  it  also  exhibited  differences  in 
its  morphology,  "instead  of  isolated  diplococci  and  strepto- 
cocci, large  masses  of  cocci  and  diplococci  were  found,  and 
forms  dividing  into  tetrads  were  common."  Nevertheless  this 
avirulent  saprophytic  pneumococcus  could,  by  a  single  "  pas- 
sage" through  a  rabbit,  be  converted  into  a  typical  parasitic 
pneumococcus  of  high  virulence.  The  occurrence  of  such  a 
remarkable  transition  would  be  regarded  as  more  significant 
if  it  were  not  that  both  organisms  bear  the  same  name  and 
are  considered — in  spite  of  the  many  differences  existing  be- 
tween them — to  be  variants  of  each  other. 

If  we  consider  the  possibility  of  a  similar  transition  in  the 
case  of  two  races  of  bacteria  less  closely  associated  with  each 
other,  we  find  little  direct  evidence  in  proof  of  its  occurrence 
— and  this  often  of  doubtful  value — but  a  great  deal  of  cir- 
cumstantial evidence  in  favour  of  the  supposition  that  it  may 
occur.  We  have  discussed  at  length  (Chapter  VIII)  such  a 
possibility  in  the  case  of  organisms  found  in  close  association 
in  the  body,  such  as  Hofmann's  bacillus  and  the  Klebs-Loeffler 
bacillus,  Staphylococcus  epidermidis  and  Staphylococcus  pyo- 
genes,  Micrococcus  catarrhalis  and  the  meningococcus,  and 
others. 

Finally,  we  have  discussed  in  detail  (Chapter  IX)  the  re- 
cords of  certain  experiments  in  the  course  of  which  bacteria 
became  so  changed  in  character  as  to  suggest  that  they  had 
undergone  transmutation. 

In  the  first  series  of  experiments — those  of  Major  Hor- 
rocks — the  results  seem  to  be  capable  of  explanation  on  other 
grounds.  In  the  first  place  adequate  precautions  do  not  appear 
to  have  been  taken  to  guarantee  the  purity  of  the  strains  at 
different  stages  of  the  experiment.  In  the  second  place,  many 
of  the  changes  in  character  stated  to  have  been  observed  may 
be  regarded  as  examples  of  temporary  variation  only,  similar 
to  those  recorded  by  many  other  observers. 

The  second  series  of  experiments — those  of  Schmitt,  and 
of  Miihlens,  Dahm  and  FUrst,  and  of  the  writer — which 
suggest  the  occurrence  of  transmutation  between  different 


152  SUMMARY  [CH.  x 

members  of  the  paratyphoid  group  of  bacilli,  are  open  to  the 
same  criticism.  In  the  first  place,  a  temporary  variation  in  one 
character  alone — namely  in  agglutination  properties — would 
sufficiently  explain  the  results  obtained.  In  the  second  place, 
these  results  may  have  been  due  to  a  secondary  invasion — in 
other  words,  it  is  conceivable  that  there  may  have  been  a  pre- 
existing but  unrecognised  infection  in  the  animals  utilised  for 
the  experiments.  This  hypothesis  we  have  shown,  from  the 
records  of  other  investigators  and  by  analogy  with  other  pro- 
cesses of  infection,  to  be  not  improbable ;  while  the  writer's 
experiments  further  demonstrate  the  ease  with  which  such  a 
secondary  invasion  may  be  overlooked. 

In  none  of  these  experiments,  therefore,  can  the  occurrence 
of  transmutation  be  regarded  as  proved,  nor,  on  close  ex- 
amination, does  its  occurrence  appear  probable. 

A  theory  which  we  propose  to  discuss  in  conclusion  suggests 
a  via  media  by  means  of  which  organisms  might  conceivably 
exchange  many  of  their  characters  and  functions  without  them- 
selves undergoing  transmutation.  This  is  the  Enzyme  theory 
of  disease. 


CHAPTER  XI 

THti  ENZYME  THEORY  OP  DISEASE 

IT  is  impossible  to  leave  this  subject  without  some  further 
mention  of  a  theory,  to  which  passing  reference  has  already 
been  made  more  than  once  in  the  foregoing  pages,  namely  the 
Enzyme  theory  of  disease. 

This  theory  predicates  that  the  results  which  follow,  and  are 
regarded  as  characteristic  of,  infection  by  a  certain  organism 
— including  both  the  pathological  lesions  produced  and  the 
train  of  symptoms  observed  clinically — are  caused  not  only 
(if  at  all)  by  the  activities  of  the  micro-organism  itself,  but 
by  the  activities  of  ultra  microscopic  bodies  of  the  nature  of 
enzymes  which  are  associated  in  each  case  with  a  particular 
bacterial  cell  in  the  same  way  that  the  ferments  of  yeast  are 
associated  with  a  particular  vegetable  cell. 

If  the  scattered  references  to  this  theory  in  the  foregoing 
pages  be  collected  together  they  will  be  found  to  constitute 
a  by  no  means  negligible  weight  of  evidence  in  favour  of  it. 
The  considerations  which  lend  support  to  the  theory  are  the 
following. 

1.  In  the  first  place,  there  is  the  observation  that  a  sapro- 
phy tic  organism  incapable  at  one  time  of  giving  rise  to  disease, 
even  after  it  has  invaded  the  living  tissues,  may  suddenly  ac- 
quire pathogenic  powers  and  give  rise  in  the  living  body  to 
definite  lesions  and  a  definite  group  of  symptoms.  Harmless 
organisms  such  as  the  saprophytic  pneumococcus,  the  Micro- 
coccus  catarrhalis  and  B.  coli,  for  example,  may  mysteriously 
acquire  the  power  to  produce  respectively  pneumonia,  menin- 
gitis and  enteric  fever.  On  the  other  hand,  virulent  pathogenic 
organisms  such  as  the  Klebs-Loeffler  bacillus,  the  meningo- 
coccus  and  B.  typhosus  may  as  mysteriously  become  deprived 
of  their  power  to  produce  respectively  diphtheria,  meningitis 


154          THE  ENZYME  THEORY  OF  DISEASE    [CH.  xi 

and  typhoid  fever.  Eyre  and  Washbourn  (1899)  showed  in  the 
case  of  the  pneumococcus  that  such  an  alteration  in  character 
could  be  brought  about,  in  one  direction,  by  a  single  passage 
through  an  animal  and  the  reverse  change  with  almost  equal 
facility.  We  have  no  explanation  of  the  processes  upon  which 
such  changes  in  character  depend  but  we  know  that  many  of 
the  conditions  which  bring  them  about  are  precisely  those 
which  foster  or  destroy  other  properties  in  organisms  which 
we  believe  to  depend  on  ferment  action  (vide  infra). 

2.  In  the  second  place,  there  is  the  observation  that  the 
pathological  lesions  and  clinical  symptoms  resulting  from,  and 
characteristic  of,  infection  by  a  certain  organism  may  be  faith- 
fully reproduced  as  a  result  of  infection  by  a  totally  different 
organism.  For  example,  we  have  noted  (vide  pp.  99  et  seq.) 
some  of  the  lesions  and  symptoms  of  diphtheria  to  be  caused 
by  the  pneumococcus,  those  of  scarlet  fever  and  of  influenza 
by  M.  catarrhalis,  those  of  cerebrospinal  fever  by  the  Klebs- 
Loeffler  bacillus,  by  M.  catarrhalis  and  by  B.  typhosus,  and 
those  of  rabies  by  the  Klebs-Loeffler  bacillus. 

The  description  of  the  last  example  given — a  case  of 
rabies  due  to  infection  by  the  bacillus  of  diphtheria— will 
bear  repetition.  It  was  recorded  by  Head  and  Wilson  (1899). 
The  diagnosis  of  rabies  was  founded  on  the  history  and 
clinical  symptoms.  "  The  well  authenticated  history  of  a  bite 
on  the  cheek  by  an  animal,  the  two  months'  incubation 
period,  the  onset  with  extreme  pain  and  numbness  in  the 
region  of  the  scar,  the  development  of  the  characteristic 
laryngeal  and  respiratory  spasms  on  attempting  to  take 
liquids,  the  spasm  at  first  being  slight  but  later  more  pro- 
nounced and  towards  the  close  again  feeble  or  absent,  the 
insomnia,  the  absence  in  the  beginning  of  fever  which  later 
in  the  illness  became  pronounced,  the  rapid  pulse  at  all 
stages,  the  attacks  of  violent  delirium  interspersed  with 
periods  of  calm  and  complete  rationality,  the  absence  of  all 
symptoms  pointing  towards  any  other  simulating  disease  and 
the  fatal  termination — all  serve  to  make  an  almost  complete 
picture  of  rabies. "  The  Klebs-Loeffler  bacillus  was  isolated 
from  the  ventricular  fluid  and  detected  in  the  nerve  cells  of 


CH.  xi]    THE  ENZYME  THEORY  OF  DISEASE          155 

the  medulla.  The  recognition  of  this  organism  was  complete 
and  beyond  doubt.  "Not  less  suggestive  of  rabies  than  the 
clinical  history  were  the  results  of  subdural  inoculations  in 
rabbits  with  emulsions  prepared  from  the  medulla  of  the 
patient.  There  occurred  the  long  period  of  incubation  (20 
and  21  days)  followed  by  phenomena  similar  to  those  in 
experimental  rabies  of  rabbits,  and  other  rabbits  inoculated 
subdurally  with  the  medulla  of  the  first  rabbits  behaved  in  a 
similar  manner."  B.  diphtheriae  was  demonstrated  after 
death  in  the  medulla  of  the  rabbits.  By  a  thorough  investiga- 
tion, full  details  of  which  are  given,  infection  by  the  virus  of 
rabies  was  definitely  excluded. 

Such  phenomena  become  intelligible  on  the  supposition 
that  both  the  lesions  and  the  symptoms  of  a  disease  result 
from  the  activity  of  particular  enzymes  which  are  usually 
associated  with  one  particular  organism  but  are  capable  of 
being  associated,  under  certain  conditions,  with  an  altogether 
different  organism. 

3.  In  the  third  place,  representatives  of  one  specific 
organism,  morphologically  and  culturally  indistinguishable 
from  one  another,  may  give  rise  in  the  living  body  to  entirely 
different  lesions  and  symptoms.  Indeed,  the  contrast  between 
the  train  of  lesions  and  symptoms  produced  in  one  case  and 
that  produced  in  another  may  be  as  marked  as  the  contrast 
between  the  lesions  and  symptoms  produced  by  two  organisms 
representing  two  distinct  species. 

For  example,  different  epidemics  of  the  same  disease  may 
present  altogether  different  features.  Thus,  strains  of  B. 
influenzae,  morphologically  and  culturally  indistinguishable 
from  one  another,  may  give  rise  to  epidemics  of  "  influenza  " 
characterised  by  symptoms  resembling  in  one  epidemic  a 
simple  coryza,  in  another  epidemic  rheumatic  fever,  in  a  third 
typhoid  fever,  and  in  a  fourth  cerebrospinal  meningitis. 

Not  only  do  different  epidemics  present  different  types  of 
disease  but  individual  cases  occurring  in  the  course  of  one 
and  the  same  epidemic,  and  undoubtedly  due  to  infection  by 
the  same  organism,  may  exhibit  a  totally  different  train  of 
symptoms.  We  have  mentioned  elsewhere,  the  account  given 


156          THE  ENZYME  THEORY  OF  DISEASE     [CH.  xi 

by  Andre wes  and  Horder  (1906)  of  a  number  of  cases  of 
contagious  disease,  obviously  passed  on  from  one  patient  to 
another,  of  which  some  presented  the  symptoms  of  scarlet 
fever  and  others  those  of  puerperal  fever  (vide  p.  98). 

Another  remarkable  instance  (recorded  by  Dunn  and 
Gordon,  1905)  has  been  already  alluded  to  but  is  of  sufficient 
interest,  in  this  connection,  to  warrant  a  second  description. 
They  mention  an  epidemic  in  Hertfordshire  characterised  by 
an  extraordinary  diversity  of  symptoms  in  different  patients. 
In  some  cases  there  were  sneezing,  coryza  and  the  ordinary 
symptoms  of  a  common  cold.  In  other  cases  patients  com- 
plained of  aches  and  pains  all  over  and  stiff  neck,  and  suffered 
subsequently  from  great  debility  ;  such  cases  had  all  the 
appearance  of  influenza.  In  others,  again,  the  illness  closely 
resembled  scarlet  fever ;  it  began  with  sore  throat,  rigors, 
vomiting,  headache,  fever  and  rapid  pulse,  and  was  ac- 
companied by  a  punctate  rash  at  the  end  of  the  first  24  hours 
(followed  later  by  desquamation),  the  "strawberry"  tongue, 
circum-oral  pallor,  enlarged  cervical  glands  which  in  some 
cases  suppurated,  and  in  some  patients  by  complications  such 
as  nephritis,  arthritis  and  otorrhoea.  A  fourth  type  resembled 
diphtheria  and  exhibited  a  suspicious  membrane  on  the 
tonsil.  A  fifth  type  was  notified  in  some  cases  as  typhoid 
fever  and  was  characterised  by  epistaxis,  melaena,  prostration 
and,  in  some  cases,  it  is  stated,  a  positive  Widal  reaction. 
Finally,  a  number  of  cases,  particularly  amongst  children, 
resembled  cerebrospinal  fever  and  were  so  diagnosed  ;  these 
were  characterised  by  profuse  nasal  discharge,  pain  in  the 
back  of  the  neck,  headache,  photophobia  and  irritability, 
dilatation  of  one  or  both  pupils,  persistent  vomiting,  drowsiness, 
head  retraction,  paralysis,  coma,  arid  sometimes  convulsions 
and  death. 

Sometimes  these  widely  divergent  types  were  exhibited  by 
the  different  members  of  a  single  family  or  household  struck 
down  by  the  disease,  either  simultaneously  or  consecutively. 
After  a  thorough  investigation,  these  observers  were  convinced 
that  the  outbreak  of  these  various  types  of  illness  was  due  to 
the  prevalence  and  spread  of  only  one  disease  and  not  a 


CH.  xi]    THE  ENZYME  THEORY  OF  DISEASE          157 

number  of  different  diseases,  and  a  bacteriological  examination 
of  a  large  number  of  cases  by  Gordon  showed  that  the  disease 
was  due  to  infection  by  an  organism  closely  resembling,  if 
not  identical  with,  M.  catarrhalis. 

Such  contrasting  groups  of  symptoms  inevitably  suggest 
to  our  minds  that  something  beside  the  mere  presence  of  the 
organism  is  responsible  for  them. 

4.  In  the  fourth  place,  one  can  trace  a  remarkable 
resemblance  between  the  conditions  which  influence  the 
development  and  the  loss  of  pathogenic  power  on  the  part  of 
micro-organisms  and  the  conditions  which  influence  the 
development  and  the  loss  of  their  power  to  ferment  carbohy- 
drates. 

(a)  The  addition,  in  small  quantities,  of  an  antiseptic — 
such  as  carbolic  acid — to  the  culture  medium  deprives  organ- 
isms growing  in  it  of  virulence  (vide  p.  75).   The  same  agency 
will  destroy  the  power  of  organisms  to  ferment  carbohydrates 
(vide  p.  55). 

(b)  The  influence  of  oxygen.   Pasteur,  30  years  ago,  found 
that  the  virulence  of  the  organism  of  chicken  cholera  was 
better  maintained  in  the  absence  of   oxygen.     Anaerobic 
growth  similarly  increases  the  virulence  of  the  cholera  spirillum 
(Hueppe,  quoted  Adami,  1892).   On  the  other  hand,  B.  diph- 
theriae  and  other  organisms  become  less  toxic  if  deprived 
of  oxygen.  The  same  factor  influences  the  activity  of  ferments. 
In  some  cases  the  absence  of  oxygen  inhibits  their  functions, 
in  other  cases  it  appears  to  augment  them.   This  is  exemplified 
by  the  sugar  splitting  ferments  associated  with  bacteria.   For 
example,  anaerobic  growth  may  increase  the  power  of  the 
dysentery  bacillus  to  ferment  maltose  (Torrey,  1905).  Andre  wes 
and  Border  (1906)  mention  a  strain  of  streptococcus  which 
failed  to  ferment  lactose  under  ordinary  conditions  but  did 
so  readily  when  deprived  of  oxygen. 

(c)  Changes    in    temperature.     It    is    characteristic    of 
enzymes  that  each  one  has  an  optimum  temperature  at  which 
its  activities  are  most  effective  and  also  higher  and  lower 
limits  of  temperature  beyond  which  its  activities  altogether 
cease.   The  digestive  enzymes  in  man  act  most  rapidly  at  the 


158          THE  ENZYME  THEORY  OF  DISEASE     [CH.  xi 

temperature  of  the  human  body — those  of  cold  blooded  animals 
at  much  lower  temperatures.  The  diastatic  ferment  of  germ 
barley  is  most  effective  at  60°  C. — a  temperature  at  which 
most  enzymes  are  destroyed.  The  phosphorescence  sometimes 
observed  in  sea- water  is  produced  by  the  Micrococcus  phos- 
phorescens  through  the  agency  of  an  enzyme  the  optimum 
temperature  of  which  is  that  of  the  sea. 

The  enzymes  which  are  associated  with  bacteria  and  bring 
about  the  fermentation  of  carbohydrates  show  a  similar 
behaviour.  We  find  that  a  strain  of  bacteria  which  will 
ferment  a  certain  "  sugar  "  at  one  temperature  will  not  do  so 
at  another.  For  example,  Wilson  (1910)  describes  a  strain  of 
B.  typhosus  which  at  22°  C.  would  ferment  lactose  within  two 
days  but  at  37°  C.  failed  to  do  so  in  a  month.  Coplans  (1909) 
observed  certain  strains  of  B.  coli  which  exhibited  the  reverse 
phenomenon,  fermenting  dulcite  more  readily  at  37°  C.  than 
at  20°  C. 

The  property  of  virulence  in  pathogenic  bacteria  is  like- 
wise governed  by  temperature.  Organisms  which  are  virulent 
when  growing  at  one  temperature  lose  their  virulence  when 
grown  at  another.  For  example,  B.  diphtheriae,  B.  tetani, 
B.  anthracis  and  many  others  (vide  p.  74)  lose  their  virulence 
at  temperatures  much  above  that  of  the  body.  The  fact  that 
no  bacterial  disease  in  cold  blooded  animals  is  communicable 
to  man  may  possibly  be  explained  on  such  grounds. 

Furthermore,  just  as  the  enzymes  which  ferment  carbo- 
hydrates are  destroyed  at  temperatures  much  above  60°  C.,  so 
we  find  the  property  of  virulence  may  be  completely  removed 
by  subjecting  an  organism  to  high  temperatures,  even  though 
the  organism  itself  survives.  The  tetanus  bacillus  is  deprived 
altogether  of  toxicity  by  growth  at  65°  C.  for  one  hour  (Muir 
and  Ritchie). 

(d)  Exposure  to  sunlight  is  another  factor  which  influences 
both  the  fermenting  power  and  the  virulence  of  organisms. 

(e)  Finally  symbiosis  is  not  without  influence.   Diseased 
conditions  may  result  from  a  mixed  infection  which  neither 
of  the  organisms  concerned  is  capable  of  producing  alone.   It 
is  said  that  a  dog  will  not  succumb  to  the  infection  of  tetanus 


CH.  xi]    THE  ENZYME  THEORY  OF  DISEASE          159 

unless  it  is  infected  simultaneously  with  pyogenic  cocci.  In 
the  same  way,  processes  of  fermentation  may  be  brought 
about  by  two  different  organisms  growing  together  in  a 
certain  medium  which  neither  can  accomplish  by  itself.  For 
example,  neither  B.  coli  nor  B.  dentrificans  alone  can  reduce 
nitrates  but  if  allowed  to  act  upon  sodium  nitrate  together 
they  bring  about  the  escape  of  free  nitrogen. 

5.  There  is,  furthermore,  a  remarkable  correspondence 
between  the  acquirement  of  virulence  by  "animal  passage" 
and  the  acquisition  of  fresh  fermenting  properties  by  prolonged 
growth  in  a  medium  containing  a  particular  sugar  : 

(a)  The  virulence  acquired  by  "passage"  through  a  certain 
animal  applies  to  that  particular  species  of  animal ;  virulence 
towards  another  species  may  be  increased  at  the  same  time 
but  towards  a  third  species  it  may  actually  be  diminished.  The 
fresh  fermenting  power  resulting  from  prolonged  growth  in  a 
sugar  concerns  that  particular  sugar ;    the  capacity  of  the 
organism  to  ferment  another  sugar  may  be  increased  simul- 
taneously while  in  respect  to  a  third  sugar  the  fermenting 
power  may  be  diminished. 

(b)  The  method  of  "passage"  is  more  effective  in  con- 
ferring virulence  if  repeated  inoculations  are  made  through  a 
series  of  animals  at  short  intervals.   The  prolonged  growth  in 
a  particular  sugar  is  more  successful  in  developing  fermenting 
power  if  repeated  subcultures  are  made  at  frequent  intervals 
on  to  media  containing  the  sugar. 

(c)  If  virulence  is  readily  acquired  on  "passage"  it  is 
easily  maintained  and  is  found  to  persist  for  a  long  time  on 
artificial  media ;    on  the  other  hand  if  it  is  very  slowly 
developed  by  "passage"  it  is  quickly  lost  outside  the  body. 
It  is  so,  also,  as  regards  fermenting  power.  In  cases  where 
the  property  is  rapidly  developed,  byx  growth  on  a  particular 
sugar,  it  is  retained  for  long  periods  on  ordinary  media ;  on 
the  other  hand,  where  it  is  very  slowly  acquired  it  is  found 
that  a  return  to  ordinary  media  is  soon  followed  by  reversion 
in  character. 

(d)  Where  virulence  has  been  lost  only  for  a  short  time 
by  a  strain  of  organisms  it  is  quickly  restored  by  "  passage  "  ; 


160         THE  ENZYME  THEORY  OF  DISEASE     [CH.  xi 

the  power  to  ferment  a  particular  sugar,  if  it  has  only  recently 
failed,  is  rapidly  regained  in  the  presence  of  that  sugar. 

6.  Bacterial  toxins,  again,  are  considered  to  be  of  two 
kinds — extra-cellular  toxins,  secreted  by  the  bacterial  cell 
into  the  surrounding  medium,  and  intra-cellular  toxins  elabor- 
ated within  the  body  of  the  cell  and  liberated  only  when  the 
cell  itself  is  disintegrated.  The  same  may  be  said  of  the 
enzymes  which  ferment  carbohydrates.   The  ferment  of  yeast, 
"invertin,"  which  transforms  cane  sugar  into  dextrose  and 
levulose,  can  be  separated  from  the  yeast  cell.   The  breaking 
up  of  the  dextrose  into  alcohol  and  other  products  is  a 
property  of  the  yeast  cell  itself  and  the  ferment  responsible 
for  this  second  stage  can  only  be  extracted  when  the  actual 
cell  body  is  expressed  (S.  Martin,  1904).  The  ferments  of  the 
alimentary  canal  may  be  distinguished  from  each  other  in  the 
same  way.   One  stage  in  digestion  is  brought  about  in  the 
lumen  of  the  intestine  by  extra-cellular  ferments  present  in 
the  secretions.   Another  stage  is  effected  within  the  actual 
cells  of  the  intestinal  wall  by  intra-cellular  ferments  acting 
upon  the  foodstuffs  as  they  are  absorbed. 

An  emulsion  of  even  a  small  portion  of  a  glandular  organ 
may  possess  far  more  power  than  its  actual  secretion,  for  the 
former  contains  the  intra-cellular  as  well  as  the  extra- cellular 
enzymes.  An  emulsion  of  pathogenic  bacteria  is  likewise  far 
more  potent  than  a  culture  containing  the  same  number  of 
organisms.  For  example,  the  smallest  fatal  dose  to  a  bovine 
animal  of  a  culture  of  tubercle  bacilli  contains  20,000  million 
organisms.  The  smallest  fatal  dose  of  an  emulsion  of  the 
bacilli  contains  only  5000  (Report  of  English  Tuberculosis 
Commission). 

7.  In  the  seventh  place,   it  may  be  observed  that  the 
property  of  virulence  is  in  many  instances  associated  with 
the  power  of  producing  fermentation.   If  we  study  two  closely 
allied  organisms,  one  of  them  virulent  and  the  other  non- 
virulent,  the  former  will  often  be  found  to  be  the  sugar 
fermenter  while  the  latter  has  no   action   in   this  respect. 
For  example,  the  Micrococcus  catarrhalis  is  comparatively 
non-virulent  and  ferments  no  sugars ;   the  gonococcus  and 


CH.  xi]    THE  ENZYME  THEORY  OF  DISEASE          161 

meningococcus  are  virulent  and  ferment  sugars.  Again, 
Hofmann's  bacillus  is  non-virulent  and  non-fermenting  while 
the  Klebs-Loeffler  bacillus  is  virulent  and  ferments.  B.  coli 
communis  is  a  sugar  fermenter  and  readily  acquires  virulence. 
We  may  explain  the  association  between  the  two  properties 
on  the  ground  that  both  are  examples  of  adaptation  and  that 
an  organism  which  possesses  unusual  power  of  adaptability 
in  one  particular  direction  may  be  expected  to  show  a  similar 
power  of  adaptability  in  another  direction  ;  but  the  associa- 
tion between  virulence  and  fermenting  power  lends  some 
support  to  the  supposition  that  the  former  may  depend  upon 
a  process  which  we  have  every  reason  to  believe  is  responsible 
for  the  latter,  namely  ferment  action. 

8.  Bacterial  invasion  is  met,  on  the  part  of  the  body,  by 
measures  calculated  to  destroy  the  organisms  and  to  counter- 
act their  toxins.  These  measures  consist  in  the  elaboration, 
by  the  fixed  cells  of  the  body  as  well  as  by  the  leucocytes,  of 
various  enzymes  (Osier  and  McCrae).   The  class  of  weapon 
forged  by  the  tissue  cells  for  purposes  of  defence  might, 
perhaps,  be  thought  to  give  some  indication  as  to  the  class  of 
weapon  it  is  designed  to  meet. 

9.  Many  other  functions  of  bacteria,  besides  the  fermen- 
tation of  carbohydrates,  are  attributed  to  ferment  action  ;  for 
example,  the  formation  of  indol,  the  coagulation  of  milk 
(Savage,  1910),  the  liquefaction  of  gelatin,  the  production  of 
pigment  (Adami)  and  the  development  of  agglutinins  (Du- 
claux).   Moreover  these  other  functions  of  bacteria,  like  their 
power  of  fermenting  carbohydrates,  appear  to  be  governed  in 
many  instances  by  the  same  conditions  which  we  have  already 
mentioned  as  influencing  their  virulence.   Thus,  the  presence 
or  absence  of  oxygen,  high  and  low  temperatures,  exposure 
to  and  protection  from  sunlight,  the  presence  of  antiseptics, 
are  all  conditions  which  markedly  aflect  the  production  of 
pigment  by  bacteria  (Adami,  1892). 

10.  Many  of  these  ferments  are  separable  from  the  bac- 
teria with  which  they  are  associated.   Twenty-five  years  ago 
it  was  proved  (Bitter,  1887,  quoted  Wood)  that  the  lique- 
faction of  gelatin  by  bacteria  was  due  to  a  ferment  which 

D.  11 


162          THE  ENZYME  THEORY  OF  DISEASE    [CH.  xi 

was  independent  of  the  bacteria  and  survived  when  the 
latter  were  killed  by  subjection  to  a  temperature  of  60°C. 
Sortinin  (ibid.)  showed  that  a  culture  fluid  after  it  had 
been  passed  through  a  Chamberland  filter,  which  removed 
all  the  bacteria,  still  retained  the  power  to  liquefy  gelatin. 
Brunton  and  McFadyean  (1889)  found  that  the  gelatin  lique- 
fying ferment  could  be  isolated  by  suitable  solvents,  in  the 
same  way  that  the  inverting  ferment  of  yeast  can  be  extracted 
with  ether. 

11.  Instances  may  be  cited  of  chemical  processes,  taking 
place  in  the  body  fluids,  which  are  invariably  associated  with 
the  presence  of  certain  micro-organisms  but  which  neverthe- 
less have  been  proved  to  be  brought  about  by  the  activity 
not  of  the  organisms  in  question  but  of  ferments  associated 
with  these  organisms  and  yet  separable  from  them.   Such  an 
instance  is  to  be  found  in  the  action  of  the  micrococcus  ureae. 
In  the  presence  of  this  organism  the  urea  of  the  urine  is  split 
up  with  the  formation  of  ammonium    carbonate.    In  the 
absence  of  this  organism  the  process  does  not  take  place  and 
if  the  process  has  begun  the  removal  of  the  organism  at 
once  stops  it.  An  ethereal  extract,  however,  of  the  micrococcus 
ureae  has  the  power  of  accomplishing  all  that  the  presence  of 
the  organism  itself  can  effect  in  this  direction.   In  other  words, 
the  results  brought  about  by  its  presence  are  due  not  to  its 
own  activities  but  to  those  of  a  ferment  "  urase  "  which  is  in 
some  way  associated  with  it  but  which  can  be  dissociated 
from  it  without  any  loss  of  function. 

12.  If  it  were  possible  to  discover  a  parallel  instance  of 
dissociation,  not  between  an  organism  and  its  chemical  func- 
tions but  between  an  organism  and  its  pathological  functions, 
the  discovery  would  give  great  weight  to  the  theory  we  are 
discussing.   Now  such  a  parallel  can  actually  be  traced  in  the 
action  of  the  pneumococcus.   Rosenow  (1912—13)  has  recently 
shown  that  the  artificial  injection  into  the  body  of  the  toxins 
manufactured  by  the  pneumococcus  may   bring  about  the 
death  of  an  animal  in  one  of  two  ways.   It  may  produce  an 
acute  bronchial  spasm  which  proves  fatal  in  a  few  hours.   If 
the  dose  of  the  toxin  is  small  the  bronchial  spasm  may  not 


CH.  xi]    THE  ENZYME  THEORY  OF  DISEASE          163 

be  sufficiently  acute  to  cause  death  and  other  symptoms  and 
lesions  follow  which  result  in  the  death  of  the  animal  in  a  few 
days.  He  discovered  that  the  particular  constituent  of  the 
toxin  responsible  for  this  bronchial  spasm  could  be  removed 
altogether  from  a  suspension  of  the  organism  by  the  addition 
of  blood  charcoal,  so  that  the  subsequent  injection  of  the 
filtered  fluid  failed  to  cause  the  bronchial  spasm,  although  it 
still  produced  the  other  symptoms  and  lesions  and  led  to  a 
fatal  termination  in  a  few  days.  He,  likewise,  discovered  that 
the  same  constituent  of  the  toxin  could — like  the  sugar- 
splitting  ferment  of  the  yeast  cell  and  the  urea-splitting 
ferment  of  M.  ureae — be  extracted  with  ether  and,  further, 
that  if  a  normal  saline  solution,  to  which  this  ethereal  extract 
had  been  added,  were  injected  into  an  animal,  the  typical 
bronchial  spasm  was  developed  in  the  complete  absence  of 
the  organism  itself. 

The  force  of  this  analogy  is  somewhat  weakened  by  the 
knowledge  that  this  acute  bronchial  spasm  is  by  no  means 
pathognomic  of  the  pneumococcus,  many  poisons  producing 
the  same  result  on  injection  into  an  animal.  The  analogy  is, 
however,  suggestive. 

13.  The  dissociation  brought  about  artificially  in  the 
laboratory  by  this  investigator  may  be  observed  to  take 
place  naturally  in  response  to  certain  kinds  of  environment. 
Thus,  a  strain  of  pathogenic  bacteria  may  lose  its  power  to 
produce  a  certain  lesion  or  to  cause  a  certain  symptom  in  the 
body.  Further,  the  conditions  which  appear  to  deprive  it  of 
such  functions  are  comparable  to  those  which,  we  have  seen, 
influence  ferment  activity.  A  few  examples  will  suffice. 

(a)  The  quality  of  light  to  which  a  culture  of  bacteria  is 
exposed  may  modify  their  power  to  produce  pigment.   Ex- 
posure to  the  ultra-violet  rays  is  found  to  alter  profoundly 
the  lesions  and  symptoms  caused  by  B.  anthracis  (Henri, 
1914). 

(b)  The  presence  or  absence  of  oxygen  influences  pigment 
production  and  the  fermentation  of  sugars  by  bacteria.   Foa 
^1890)  isolated  strains  of  pneumococci  from  the  lung  and  from 
the  spinal  fluid  of  a  rabbit  which  had  died  after  inoculation 

11—2 


164          THE  ENZYME  THEORY  OF  DISEASE     [CH.  xi 

with  this  organism.  The  strain  from  the  lung  possessed  the 
property  of  causing,  when  inoculated  into  another  rabbit,  an 
inflammatory  oedema  of  the  skin  ;  the  strain  from  the  spinal 
fluid  failed  to  do  so.  The  strain  from  the  lung,  however,  when 
grown  anaerobically  was  deprived  of  its  power  to  cause  this 
inflammatory  oedema  of  the  skin. 

(c)  Growth  in  a  certain  vehicle  may  alter  the  fermenting 
powers  of  one  organism  and  the  pathogenic  powers  of  another. 
The  fermentation  properties  of  a  strain  of  B.  coli  isolated  from 
cowdung  become  altered  after  growth  in  milk.   A  milk-borne 
epidemic  of  scarlet  fever  is  not  infrequently  characterised  by 
the  partial  or  complete  absence  of  the  usual  rash. 

(d)  An  analogy  may  also  be  traced  between  the  action  of 
chemical  substances  added  to  culture  media  and  the  effects 
of  drugs  administered  in  disease.  For  example,  the  presence 
of  sodium  benzoate  inhibits  the  power  of  B.  coli  to  produce 
gas  from  dextrose — one  of  the  most  stable  and  fundamental 
differences  separating  B.  coli  from  the  typhoid-dysentery 
group — without  in  any  way  affecting  its  other  fermenting 
reactions.   The  administration  of  sodium  salicylate  in  rheu- 
matic fever  eliminates  the  symptoms  of  pain  and  fever — 
the  two  most  characteristic  symptoms  of  this  disease — with- 
out apparently  affecting  any   other  of  its   symptoms  and 
lesions  in  a  great  many  cases. 

14.  If  the  foregoing  considerations  suggest  that  the 
symptoms  of  disease  are  due  to  zymotic  action  they  likewise 
imply  that  each  separate  symptom  is  attributable  to  the 
activity  of  a  distinct  enzyme.  Such  a  conclusion  postulates 
the  existence  of  innumerable  pathogenic  enzymes  each  one 
concerned  in  the  causation  of  some  particular  symptom  of 
disease,  and  requires  us  to  conceive  of  different  groups  or 
combinations  of  enzymes  associated  with  different  pathogenic 
organisms  and  responsible  in  the  case  of  each  organism  for  the 
train  of  symptoms  that  follow  its  invasion  of  the  living 
tissues. 

Analogy  with  the  sugar-fermenting  properties  of  bacteria 
renders  such  a  complex  picture  of  the  causation  of  disease 
less  fanciful  than,  at  first  sight,  it  appears.  As  we  have  shown 


CH.  xi]    THE  ENZYME  THEORY  OF  DISEASE          165 

(vide  p.  60)  it  can  be  proved  that  not  only  is  the  fermentation 
of  different  carbohydrates  effected  by  distinct  and  appropriate 
ferments  but  each  of  the  several  stages  in  the  fermentation 
of  a  single  carbohydrate — such  as  the  formation  of  acids  and 
the  production  from  these  acids  of  gas — is  carried  out  by  its 
distinct  and  appropriate  ferment.  Moreover  different  carbo- 
hydrates yield  on  fermentation  different  acids  and  each 
different  acid  requires  to  be  acted  on  by  a  special  ferment 
before  it  becomes  split  up  into  gaseous  products.  If  such  a 
comparatively  simple  result  as  the  production  of  acid  and 
gas  in  various  carbohydrate  media  requires  the  co-operation 
of  so  many  distinct  ferments,  the  extremely  complex  and 
diverse  results  of  the  bacterial  invasion  of  the  body  would 
appear  to  demand  proportionately  greater  complexity  and 
diversity  in  the  zymotic  agents  causing  them. 

We  have  seen  that  the  enzymes  concerned  with  the  fer- 
mentation of  particular  carbohydrates  are  definitely  associated 
with  certain  vegetable  and  bacterial  cells  but  not  with  others. 
For  example,  many  yeasts  are  able  to  invert  sugar  but  only 
three  yeasts  are  known  which  are  able  to  ferment  lactose. 
Proteolytic  ferments  are,  likewise,  associated  only  with 
certain  vegetable  cells,  such  as  the  papain.  Proteid-splitting 
and  lactose-splitting  ferments  are  associated  with  certain 
bacteria  of  the  typhoid-coli  group  but  not  with  others.  It  is 
conceivable  that,  in  precisely  the  same  way,  the  agencies 
responsible  for  certain  definite  symptoms  in  disease  might  be 
definitely  associated  with  some  bacterial  cells  but  not  with 
others. 

A  further  suggestion  occurs  to  one  at  this  point.  If  the 
enzyme  responsible  for  one  particular  symptom  of  a  disease 
can  be  dissociated  from  the  specific  organism  of  that  disease, 
should  we  not  expect  to  be  able,  by  suitable  methods,  to 
dissociate  from  the  organism  not  one  only  but  all  the  enzymes 
causing  the  various  symptoms  of  the  disease  in  question  ? 

15.  If  such  a  complete  dissociation  were  practicable  it 
should  be  possible  to  accomplish  two  things  ;  in  the  first 
place,  to  deprive  an  organism  of  its  power  to  produce  a  single 
one  of  the  symptoms  of  the  disease  associated  with  it  and,  iu 


166          THE  ENZYME  THEORY  OF  DISEASE    [CH.  xi 

the  second  place,  to  reproduce  faithfully  the  complete  train 
of  symptoms  and  lesions  characteristic  of  a  disease  in  the 
entire  absence  of  the  specific  organism  to  which  the  disease 
is  commonly  attributed.  Both  these  results  have  actually 
been  observed.  As  regards  the  first,  numerous  examples  have 
already  been  given  of  virulent  organisms,  normally  capable  of 
giving  rise  to  a  complex  and  characteristic  train  of  symptoms 
and  lesions  in  the  living  body  (e.g.  the  Klebs-Loeffler  bacillus, 
B.  typhosus}  being  deprived  of  their  power  to  produce  a 
single  one  of  these  symptoms  or  lesions  although,  in  every 
other  respect,  retaining  their  character  and  properties  un- 
changed (vide  "  Virulence,"  "  Pathogenesis  "). 

It  is  well  recognised,  for  instance,  that  infection  with  B. 
typhosus  may  occur  without  any  of  the  clinical  manifestations 
of  typhoid  fever.  Dudgeon  (1908)  quotes  three  cases  of 
patients  whose  stools  contained  enormous  numbers  of  typhoid 
bacilli  and  whose  blood  agglutinated  these  organisms  in 
dilutions  of  1  in  200,  who  nevertheless  failed  to  exhibit  a 
single  symptom  of  typhoid  fever. 

With  regard  to  the  second,  examples  may  be  cited  of 
diseases  associated  with  the  presence  of  certain  bacteria  but 
now  generally  recognised  as  being  due  to  "filter  passers." 
Hog  cholera,  for  instance,  is  a  highly  contagious  disease 
associated  with  a  certain  bacillus,  the  "  hog  cholera  bacillus." 
A  pig  suffering  from  the  disease  can  infect  other  healthy 
pigs  ;  the  latter  develop  the  same  symptoms  and  are  found  to 
be  invaded  by  the  same  organism  and  they  are  capable,  in  their 
turn,  of  infecting  other  healthy  animals  in  precisely  the  same 
way.  It  has  been  shown,  however,  that  a  broth  culture  of 
the  hog  cholera  bacillus,  from  an  infected  animal,  after  it  has 
been  passed  through  a  Chamberland  filter — a  process  which 
entirely  removes  any  bacilli  present — nevertheless  retains  its 
power  to  "infect"  a  healthy  animal  with  hog  cholera,  the 
disease  running  the  same  course  as  usual  and  exhibiting 
precisely  the  same  lesions  and  symptoms. 

Such  a  sequence  affords  a  precise  analogy  to  the  ex- 
periment of  Sortinin,  a  quarter  of  a  century  ago,  which  led  to 
his  discovery  that  after  a  culture  of  certain  bacteria  had  been 


CH.  xi]    THE  ENZYME  THEORY  OF  DISEASE          167 

passed  through  a  Chamberland  filter,  a  bacteria-free  filtrate 
was  obtained  which  nevertheless  retained  the  power  of  the 
original  culture  to  liquefy  gelatin. 

16.  One  objection  may  be  urged  at  this  point,  namely, 
that  it  has  not  hitherto   been  possible  to   separate   from 
any  pathogenic  organism  an  enzyme  capable  of  producing, 
outside  the  living  body,  the   toxins  characteristic  of  that 
organism.  It  is,  however,  equally  impossible  in  many  cases 
to   isolate    from    bacteria  agents  which    will  bring    about 
other  of  their  functions  which  we  recognise  to  depend  on 
ferment  action.   Moreover,  we  have  discussed  under  the  head 
of  virulence  (vide  p.  77)  some  of  the  qualities  in  which 
artificial  media  differ  from  the  vital  fluids  of  the  body  and 
such  differences  may  well  prove  an  insuperable  obstacle  to 
the  performance  by  an  enzyme  of  its  usual  functions.   A  pick- 
pocket may  ply  his  "trade"  vigorously  in  a  busy  crowded 
thoroughfare  and  yet  a  few  hours  later,  in  a  workhouse  ward, 
give  no  sign  of  his  peculiar  abilities.   In  the  latter  situation 
certain  things  are  lacking — the  incentive   which  normally 
stimulates  him  (that  is  to  say,  the  "struggle  for  existence"),  the 
materials  he  seeks  to  gain,  the  conditions  essential  to  his 
work — and  this  fact  may  render  difficult  if  not  impossible 
any  display  of  his  customary  activities. 

The  enzyme  theory  of  disease  is  not  at  the  present  stage 
of  our  knowledge  capable  of  proof.  The  above  considerations, 
however,  lend  some  measure,  if  not  of  certainty  at  least  of 
probability  to  the  supposition  that  the  organisms  associated 
with  certain  diseases  are  not  themselves  the  causal  agents  of 
those  diseases  but  merely  act  as  carriers  of  ultra-microscopic 
bodies,  possibly  parasitic  in  character,  which  have  hitherto 
eluded  detection  but  which  are  the  real  causal  agents  of  the 
lesions  and  symptoms  produced. 

17.  If  such  an  hypothesis  should  ultimately  prove  to  be 
correct,  how  would  it  affect  our  ideas  as  to  the  possibility  of 
transmutation  occurring  amongst  bacteria?  Obviously,  if  it  is 
possible  for  the  enzyme  or  enzymes  which  produce  a  certain 
disease  to  become  dissociated  from  the  organism  to  which  that 
disease  is  commonly  attributed  and  to  become  attached  to  some 


168          THE  ENZYME  THEORY  OF  DISEASE     [CH.  xi 

other  organism,  the  effect,  though  not  the  actual  process,  of 
transmutation  would  be  brought  about. 

A  transference  of  this  kind  would  present  certain  diffi- 
culties. The  enzymes — if  such  be  their  true  nature — of  disease 
would  appear  to  depend,  to  some  extent,  for  their  activity 
upon  the  structure  and  metabolism  of  the  cell  body  to  which 
they  are  attached  and  if  they  are  to  be  transferred  from  one 
organism  to  another  without  loss  of  function  the  second  host 
must  possess  those  characters  in  the  way  of  structure  and 
metabolism  which  are  vital  to  the  activity  of  the  enzymes. 
This  implies  certain,  and  possibly  rigid  limitations.  The  pro- 
blem can  best  be  illustrated  by  analogy  with  more  familiar 
things. 

We  are  able  to  distinguish  at  sea,  a  fleet  of  fishing  smacks, 
a  line  of  battleships,  a  couple  of  pleasure  steamers,  a  solitary 
four  masted  barque  in  full  sail.  We  distinguish  these  different 
types  of  vessels  readily  from  one  another  by  characters  analo- 
gous to  the  "  morphology "  of  bacteria,  that  is  to  say  their 
size,  shape,  motility  and  grouping.  We  have,  however,  another 
way  of  distinguishing  them,  namely  by  observing  the  effects 
produced  by  their  arrival  at  a  port,  analogous  to  the  effects 
of  bacterial  "  invasion.'3  The  arrival  of  the  fleet  of  fishing 
smacks  is  followed  by  a  rush  of  people  from  their  houses  to 
the  shore  (comparable  to  the  exudation  of  leucocytes),  a 
silvery  deposit  on  the  quay-side  as  they  empty  their  fish,  re- 
placed in  a  few  hours  by  a  brownish  membrane  as  the  nets  are 
spread  out  to  dry.  The  train  of  "  symptoms  "  is  invariable  and 
becomes  associated  in  our  minds  with  the  entry  into  port  of 
this  type  of  vessel.  So,  too,  with  the  others.  The  appearance 
of  gunboats  may  be  followed  by  the  destruction  of  a  town 
(comparable  to  necrosis).  The  arrival  of  the  pleasure  steamers 
may  be  greeted  with  a  display  of  fireworks  (comparable  to 
pyrexia),  that  of  the  tall  barque  with  its  cargo  of  spirits  may 
give  rise  to  general  intoxication  (comparable  to  delirium). 

Such  a  sequence,  however,  is  not  invariable.  For  example, 
the  fishing  smacks  might  be  employed  in  smuggling  and  land 
a  cargo  of  spirits,  giving  rise  to  intoxication  on  shore.  The 
gunboats  might  be  employed  by  Royalty  on  a  pleasure  cruise 


CH.  xi]    THE  ENZYME  THEORY  OF  DISEASE          169 

and  their  arrival  be  greeted  with  fireworks.  A  couple  of  in- 
nocent looking  steamers  might  be  engaged  in  piracy  and  open 
a  destructive  fire  from  their  guns.  The  tall  barque  might  con- 
ceivably land  a  cargo  of  fish.  In  other  words  each  type  of 
vessel  might  give  rise  to  a  train  of  events  rightly  regarded  as 
characteristic  of  an  altogether  different  type,  for  the  effects 
they  produce  depend  not  on  the  activities  of  the  ships  them- 
selves, which  are  merely  carriers,  but  on  those  of  their  occu- 
pants. 

At  the  same  time  the  function  of  each  different  type  of 
vessel,  though  dependent  upon  its  occupants,  is  also  to  some 
extent  governed  by  its  structure  and  the  equipment  it  carries 
(comparable  to  the  structure  and  metabolism  of  a  micro- 
organism). A  mere  exchange  of  crews  would  not  necessarily 
effect  an  exchange  of  function.  For  example,  a  party  of 
fishermen  sent  to  sea  in  an  ironclad  would  be  as  unlikely  to 
land  a  catch  of  fish  as  a  force  of  naval  officers  and  seamen 
embarked  in  fishing  smacks  would  be  to  bombard  a  town. 

In  one  respect  our  analogy  fails.  Hitherto  we  have  spoken 
of  the  enzyme  as  something  grafted  on  to  the  micro-organism, 
in  the  nature  of  a  parasite,  but  there  is  much  to  suggest  in 
the  evidence  we  have  quoted  that  it  is,  in  reality,  a  body  ela- 
borated by  the  organism  itself,  comparable  to  one  of  Ehrlich's 
*'  side-chains."  Such  a  conception  of  its  nature  would  go  far 
towards  explaining  the  apparent  dependence  of  the  "enzymes" 
of  a  particular  disease  upon  a  particular  organism.  But  every 
argument  in  favour  of  such  a  supposition  in  the  case  of  the 
enzymes  which  cause  disease  applies  equally  to  our  conception 
of  the  nature  of  those  which  ferment  carbohydrates.  The  pur- 
pose of  the  arguments  here  presented  has  not  been  to  explain 
the  precise  nature  of  these  ultra-microscopic  bodies  but  merely 
to  show  that  the  lesions  and  symptoms  of  disease  may  with 
some  confidence  be  attributed  to  the  action  of  the  same  class 
of  body  as  that  to  which  we  unhesitatingly  attribute  the  fer- 
mentation of  sugars. 


11—5 


CHAPTER  XII 

CONCLUSIONS 

1.  Variation  occurs  in  every  character  of  bacteria. 

2.  These  variations  may  be  either  "  spontaneous  "  or  "  im- 
pressed "  by  conditions  of  environment. 

3.  The  recognition  of  "  species  "  amongst  bacteria  must, 
therefore,  depend  upon  a  consideration  of  their  biological 
characters  as  a  whole  and  upon  the  stability  these  characters 
display. 

4.  Transmutation  differs  from  variation  in  degree  alone  ; 
it  is  a  question  of  the  extent  of  the  modification  and  the  de- 
gree of  permanence  it  exhibits. 

5.  Transmutation  differs  from  evolution  in  degree  alone  ; 
it  is  a  question  of  the  rapidity  of  the  change. 

6.  The  occurrence  of  transmutation  between  closely  allied 
organisms  in  the  human  body  is  not  capable  of  proof  but  is 
suggested  by  circumstantial  evidence. 

7.  Supposed  instances  of  transmutation,  brought  about  by 
experimental  inoculation  of  animals,  are  shown  to  rest  on  in- 
conclusive evidence. 

8.  The  Enzyme  theory  of  disease  suggests  a  means  by 
which  bacteria  may  exchange  many  of  their  characters  and 
functions  without  themselves  undergoing  transmutation. 


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1910,  vol.  xxxni,  p.  250. 


CAMBRIDGE  :     PRINTED   BY  J.    B.   PEACE,    M.A.,    AT   THE   UNIVERSITY  PRESS. 


178  APPENDIX 

SAVAGE,  W.  G.  "Gelatin  surface  colonies  of  Bacillus  colicommunis."  Journal 

of  Pathology  and  Bacteriology,  1904,  vol.  ix,  p.  347. 
Further    Report    upon  the    Presence   of  the   Gaertner  group  of 

Organisms  in  the  Animal  Intestine.    Reports  to  Local  Gov.  Board, 

vol  xxxvn,  1907-8. 
"The  coagulation  of  milk  by  Bacillus  coli  communis."    Journal  of 

Pathology  and  Bacteriology,  1905,  vol.  x,  p.  90. 
SCHMITT,  F.  M.     "Zur  Variabilitat  der  Enteritis-bakterien."  Zeitschrift  f, 

Infektionskrankheiten,  parasitare  Krankheiten  und  Hygiene    der 

Haustiere,  1911,  vol.  9,  p.  188. 
SCHOTMULLER.    See  Muir  and  Ritchie,  p.  207. 
SCHULTZ,  0.  T.     "The  Proportion  of  Granular  and  Barred  forms  of  Bacillus 

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vol.  vi,  p.  610. 
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Contribution  to  the  Study  of  the  Relationship  between  Avian  and  Human 

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1907,  p.  17. 
SMIRNOW,  M.  R.    "Some  Symbiotic  Relations  of  the  Bacillus  diphtheriae." 

Journal  of  Medical  Research,  1908,  vol.  xvm,  p.  257. 
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SMITH,  Theobald.    "The  Relation  of  Dextrose  to  the  production  of  Toxin  in 

Bouillon  Cultures  of  the  Diphtheria  bacillus."  Journal  of  Experimental 

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vol.  6,  p.  401  (Originate). 

SOUTHARD.     Boston  Med.  and  Surg.  Journal,  1910,  vol.  162,  p.  452. 
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vol.  ix,  p.  10. 
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vol.  xxxvi,  p.  307,  Referate. 
THIELE,  F.  H.  and  EMBLETON,  D.    "The  Pathogenicity  and  Virulence  of 

Bacteria."  Journal  of  State  Medicine,  1914,  May,  p.  270. 
THOMSON,  W.  H.     "Vagaries  of  the  Pneumococcus."    The  Medical  Record, 

New  York,  1911,  vol.  LXXIX,  p.  565. 

TORREY,  J.  C.     "A  Comparative  Study  of  Dysentery  and  Dysentery-like  Or- 
ganisms." Journal  of  Experimental  Medicine,  1905,  vol.  vn,No.  4,  p.  365. 
TWORT,  F.  W.     "The  Fermentation  of  Glucosides  by  Bacteria  of  the  Typhoid 

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Series  B,  vol.  LXXIX,  p.  329. 
WALKER,  E.  W.  Ainley.  "On  Variation  and  Adaption  in  Bacteria.  Illustrated 

by  observations  upon  Streptococci  with  special  reference  to  the  value 


APPENDIX  179 

of  Fermentation  Tests  as  applied  to  these."    Proc.  of  Royal  Society, 

1911,  Series  B.  vol.  LXXXIII,  p.  541. 
WALKER,  E.  W.  Ainley.  "Immunisation  against  Immune  Serum."  Journal  of 

Pathology  and  Bacteriology,  1903,  vol.  vm,  p.  34. 
and  MURRAY,   W.    "The  effect  of  certain  Dyes  upen  the  Cultural 

Characters  of  the  Bac.  typhosus  and  some  other  Organisms."   Brit.  Med. 

Journ.,  1904,  vol.  n,  p.  16. 
WASSERZUG,  E.    "Variations  de  forme  chez  les  bacteries."   Annales  de 

VInstitut  Pasteur,  1888,  u,  p.  75. 
WARD,  Marshall,  and  BLACKMAN.    Encyclopedia  Britannica,  xith  Edition, 

1910.    (Article  "Bacteriology.") 
WESBROOK,  F.  S.,  WILSON,  F.  B.,  MCDANIEL,  0.   "Varieties  of  B.  diphtheriae." 

Trans,  of  Assoc.  of  American  Physicians,  1900,  vol.  xv,  p.  198. 
WHIPPLE,  G.  C.  and  MAYER,  A.   "On  the  Relation  between  Oxygen  in  Water 

and  the  Longevity  of  the  Typhoid  bacillus."    Journal  of  Infectious 

Diseases,  1906,  supp.  No.  2,  p.  76. 
WILLIAMS,  Anne  W.    "Persistence  of  Varieties  of  the  Bacillus  Diphtheriae 

and  of  Diphtheria-like  bacilli."  Journal  of  Medical  Research,  1902, 

vol.  vm,  p.  83. 
WILSON,  W.  J.    "Variation  among  Bacteria."    Brit.  Med.  Journ.,  1910, 

Dec.  17th. 

"Bacteriological  Observations  on  Colon  Bacilli  of  the  'Anaerogenes 

class'."   Journal  of  Hygiene,  vol.  vm,  No.  4,  Sept.  1908. 

"Pleomorphism  as  exhibited  by  Bacteria  grown  on  Media  containing 

Urea."  Journal  of  Pathology  and  Bacteriology,  1906,  vol.  xi,  p.  394. 

WINSLOW,  C.  E.  A.    "The  Systematic  Relationship  of  the  Coccaceae." 
WOOD,  G.  E.  Cartwright.    "Enzyme  Action  in  Lower  Organisms."  Proc. 

of  Royal  Soc.  Edinburgh,  1889,  vol.  xvi,  p.  27. 
ZURCH  and  WEICHEL.    Arbeiten  aus  dem  Kaiserlichen  Gesundheitsamte, 

1910,  vol.  xxxm,  p.  250. 


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